Roles for Internal and Flanking Sequences in Regulating the Activity of Mating-Type-Silencer-Associated Replication Origins in Saccharomyces cerevisiae

Corresponding author: Joel A. Huberman, Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Sts., Buffalo, NY 14263-0001., huberman{at}acsu.buffalo.edu (E-mail)

Communicating editor: J. RINE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.

IN Saccharomyces cerevisiae a group of autonomously replicating sequence (ARS) elements—DNA sequences that function as replication origins in plasmids—is located near the left end of chromosome III (Fig 1). The ARS elements in this group, ARS301–ARS304 plus ARS320, are inactive as replication origins in their normal chromosomal locations. Instead of firing on their own, they are passively replicated by the replication fork from the nearby active origin, ARS305 (DUBEY et al. 1991 ; VUJCIC et al. 1999 ).

View larger version (8K): In this window In a new window Download PPT slide   Figure 1. Locations of origins and ARS elements, and directions of replication fork movement, in the leftmost quarter of S. cerevisiae chromosome III. The thick horizontal black line with scale in kilobase pairs represents distances along chromosome III, starting at the left telomere and extending toward the centromere. The positions of two genes (HML and HIS4) are indicated for reference; most genes are not shown. The positions of ARS elements (NEWLON et al. 1991 ; VUJCIC et al. 1999 ) are indicated by small boxes below the line and by corresponding numbers above the line. The numbers corresponding to the two ARS elements that are active as replication origins (HUBERMAN et al. 1988 ; ZHU et al. 1992 ) are indicated in boldface. The directions of the replication forks emanating from the two origins (O) are indicated by the gray arrows at the bottom (HUBERMAN et al. 1988 ; DUBEY et al. 1991 ; ZHU et al. 1992 ; additional unpublished results determined by neutral-alkaline 2-D gel electrophoresis).

Two of these inactive origins, ARS301 and ARS302, are parts of the cis-acting silencer elements (HML-E and HML-I, respectively) that are essential for repressing transcription of the mating type genes in the HML mating type cassette (MAHONEY and BROACH 1989 ; MAHONEY et al. 1991 ). However, when silencing of the mating type cassettes is abrogated by mutation of the gene encoding Sir4p (one of the trans-acting proteins essential for silencing; reviewed in LAURENSON and RINE 1992 ), ARS301 and ARS302 remain inactive as origins (DUBEY et al. 1991 ). Thus, although ARS301 and ARS302 contribute to the formation of an altered chromatin structure that is essential for transcriptional silencing (reviewed in LUSTIG 1998 ; STONE and PILLUS 1998 ), their inactivity as chromosomal replication origins does not appear to be a consequence of that altered structure. Furthermore, chromosomal inactivity is not a required feature of silencer-associated ARS elements. The ARS elements associated with the HMR-E and -I silencers near the right end of chromosome III are active (though inefficient) as origins (RIVIER and RINE 1992 ; DEBEER and FOX 1999 ; HURST and RIVIER 1999 ; RIVIER et al. 1999 ).

Results from three studies (SANTOCANALE et al. 1999 ; VUJCIC et al. 1999 ; WEINBERGER et al. 1999 ) suggest that ARS301 is normally inactive because it is programmed to fire so late in S phase that it is passively replicated by the fork from early-firing ARS305 before it has a chance to fire on its own (Fig 1). ARS301 became partially active in its native chromosomal location when ARS305 and ARS306 were deleted (VUJCIC et al. 1999 ). Under these conditions, the replication fork from the closest active origin, ARS307, did not reach ARS301 until very late in S phase. The results indicate that in some cells ARS301 fired on its own before the arrival of the fork from ARS307 (VUJCIC et al. 1999 ). In an independent study, SANTOCANALE et al. 1999 attempted to activate ARS301 by using rad53 and mec1 mutant cells. Mutations in these checkpoint genes had previously been shown to accelerate the firing of late origins (SANTOCANALE and DIFFLEY 1998 ; SHIRAHIGE et al. 1998 ). When such mutant cells were treated with hydroxyurea, which permits origin firing but blocks extensive fork progression, ARS301 was partially activated (SANTOCANALE et al. 1999 ). These results suggest that the combination of accelerated firing of late origins and inhibition of replication forks from early origins permitted ARS301 to fire before being inactivated by the fork from ARS305. Similarly, activation of ARS301 was also observed (WEINBERGER et al. 1999 ) after hydroxyurea treatment in orc2 mutant cells, in which the firing of late origins is also accelerated (SHIRAHIGE et al. 1998 ). Although these studies did not directly address ARS302, their results are consistent with the hypothesis that ARS302, like ARS301, is normally inactive because it is programmed to fire in very late S phase and is passively replicated by the fork from ARS305 before it can fire on its own.

Previous investigations of the nucleotide sequences required for the function of chromosomally active origins (MARAHRENS and STILLMAN 1992 ; RAO et al. 1994 ; RASHID et al. 1994 ; THEIS and NEWLON 1994 ; HUANG and KOWALSKI 1996 ) revealed a modular structure. An essential A domain containing the 11-bp ARS consensus sequence (ACS) is flanked by a multipart B domain on the 3' side of the T-rich strand of the ACS (MARAHRENS and STILLMAN 1992 ; RAO et al. 1994 ; RASHID et al. 1994 ; THEIS and NEWLON 1994 ; HUANG and KOWALSKI 1996 ) and in some cases by a C domain on the 5' side (WALKER et al. 1990 ). The protein complex, origin recognition complex (ORC), binds to the A and the nearby B1 domains (BELL and STILLMAN 1992 ; DIFFLEY and COCKER 1992 ; RAO and STILLMAN 1995 ; ROWLEY et al. 1995 ). Whether chromosomally inactive ARS elements or silencer-associated ARS elements have a similar modular structure has not previously been tested.

In this study, we defined the minimal sequences that are required for ARS301 and ARS302 function as plasmid replication origins, and we explored the roles of internal and flanking sequences in rendering these origins inactive in their normal chromosomal locations.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and methods:
Yeast strain CT7-11 (Table 1) was used for the deletion and linker substitution analyses of ARS301 and ARS302. Two-step gene replacement (SCHERER and DAVIS 1979 ; BOEKE et al. 1987 ) was employed to insert ARS elements into new chromosomal locations in appropriate recipient strains (Table 1). Details of strain construction (SHARMA 2000 ) are available upon request.

Mutational analyses of ARS301 and ARS302:
The nucleotide sequences studied here are part of the complete nucleotide sequence of S. cerevisiae chromosome III, GenBank accession no. NC_001135. All of the constructs described here were confirmed by sequencing. Standard in vitro mutagenesis techniques were employed. Details of methodology and all primer sequences are available upon request.

The clustered point mutations introduced into ARS301 are as follows (wild-type sequence is upper case; mutations are lower case): 11/11 ACS match, TTaTATcTaTT; 10/11 ACS match, AtAtgATTAAA; Rap1p binding site, TATGAATGcGaaT. The clustered point mutations introduced into ARS302 are as follows: 11/11 ACS match, TTTgAatTcTT; 10/11 ACS match a, AAAATgaattc; 10/11 ACS match b, gAAttcAAAAT; 10/11 ACS match c, ATTgAatTcTA; Abf1p binding site, ACGTTTGaAtTcATA.

For the purpose of evaluating their replication efficiency, all mutant constructs were cloned into a plasmid, pMW311, which is a derivative of pRS306 (SIKORSKI and HIETER 1989 ) containing a centromere (CEN5) and a selectable marker (URA3).

Evaluation of replication efficiency:
Standard methods were employed for yeast transformation and growth under selective (no uracil) and nonselective (complete medium) conditions (SHERMAN 1991 ). Single transformant colonies were restreaked onto selective plates, and single colonies from the restreaking were inoculated into 3 ml selective medium and incubated for 20–24 hr at 30°. About 250 cells were then plated on nonselective and selective plates to determine the percentage of plasmid-containing cells under selection (replication efficiency). For transformants with high plasmid loss rates, more cells were plated on the selective plates to achieve 250 final colonies.

DNA isolation and two-dimensional gel analysis:
DNA was isolated from logarithmically growing cells (1–1.5 x 10⁷/ml) using the glass bead method (HUBERMAN et al. 1987 ), and the replicating DNA was enriched as described (HUBERMAN et al. 1987 ). The two-dimensional (2-D) gel analyses were as described (BREWER and FANGMAN 1987 ), as were Southern blotting, hybridization, and image processing (DUBEY et al. 1991 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We wished to determine the extent to which ARS elements that are silencer associated and inactive in chromosomes (such as ARS301 and ARS302) differ from chromosomally active nonsilencer-associated ARS elements (such as ARS1, ARS305, and ARS307). This requires identifying and comparing the sequences within each ARS element that are important for its function as a replication origin in a plasmid. The ability of an ARS element to serve as a replication origin in a plasmid is frequently referred to as its "ARS activity." Since the sequences important for the ARS activities of several chromosomally active nonsilencer-associated ARS elements were previously characterized in detail (MARAHRENS and STILLMAN 1992 ; RAO et al. 1994 ; RASHID et al. 1994 ; THEIS and NEWLON 1994 ; HUANG and KOWALSKI 1996 ), it was sufficient for us to similarly characterize the sequences needed for ARS activity by ARS301 and ARS302.

These two ARS elements were originally localized by deletion analyses (BROACH et al. 1982 ). However, the regions defined by the deletions were not tested for independent ARS activity, leaving considerable uncertainty as to the boundaries of ARS301 and ARS302. Consequently our first task was to accurately localize these ARS elements.

Preliminary experiments revealed that a PCR-derived 256-bp stretch contained full ARS301 activity (Fig 2A). This stretch contains one 11/11 and one 10/11 match to the ACS as well as a putative Rap1p binding site (BUCHMAN et al. 1988 ). Similar experiments revealed that a 275-bp HincII-HindIII restriction fragment (Fig 2B) contains the bulk of ARS302 activity. This fragment has one 11/11 match and three 10/11 matches to the ACS as well as putative Abf1p and Rap1p binding sites (BUCHMAN et al. 1988 ).

View larger version (31K): In this window In a new window Download PPT slide   Figure 2. Characterization of ARS301 and ARS302. (A) ARS301. The starting nucleotide is 11136 in GenBank NC_001135. (B) ARS302. The starting nucleotide is 14565 in GenBank NC_001135. Replication efficiency was measured as the percentage of plasmid-bearing cells under selection (MATERIALS AND METHODS). "0" indicates that the cells could not grow under selection due to total lack of plasmid replication. The results shown are an average of three independent experiments with standard deviations. The ARS305 plasmid employed as control in A, line 18, was constructed by cloning the 550-bp NruI-ClaI fragment containing ARS305 into the same centromere-bearing plasmid used to evaluate ARS301 and ARS302 activity.

ARS301 is similar to most other S. cerevisiae ARS elements:
Intact ARS301 (Fig 2A, line 1) displays high replication efficiency, which is slightly higher than that of ARS305, a well-characterized strong ARS element (HUANG and KOWALSKI 1993 , HUANG and KOWALSKI 1996 ; LIN and KOWALSKI 1997 ; Fig 2A, line 18). To identify the essential ACS match and to assess the contribution of the putative Rap1p binding site to ARS301 function, we generated clustered point mutations targeting the ACS matches and the Rap1p binding site (Fig 2A, lines 2–4). Mutation of the 11/11 ACS match abolished ARS activity, but mutation of the 10/11 match had no effect. Thus ARS301, like most other tested ARS elements, has only one essential ACS match. Mutation of the Rap1p binding site reduced ARS activity only slightly.

Progressive deletions from either side of the essential 11/11 ACS within the 256-bp stretch were generated by PCR, and each deletion was tested for ARS activity as above. Deletion of up to 60 bp from the 3' side (Fig 2A, lines 10–12) did not greatly reduce ARS activity, but further deletions (lines 13–15) had significant effects. In contrast, none of the deletions 5' of the ACS caused an appreciable change in ARS activity (Fig 2A, lines 5–9). Consistent with the directed mutation analysis (Fig 2A, line 4), complete deletion of the Rap1p binding site had no major effect (compare lines 8 and 9 and lines 16 and 17). Combination of the 3' and 5' deletions revealed that a short stretch of DNA—95 bp—comprising an essential ACS match and 3' flanking sequences (Fig 2A, line 17) is about two-thirds as active as intact ARS301. Thus ARS301, like well-characterized chromosomally active nonsilencer-associated S. cerevisiae ARS elements (reviewed in THEIS and NEWLON 1996 ) consists primarily of an essential ACS match and 3' flanking B-domain sequences.

ARS302 requires A, B, and C domains:
Preliminary experiments (not shown) revealed that a 275-bp HincII-HindIII fragment contains the bulk of ARS302 activity. However, intact ARS302 (Fig 2B, line 1) has a much lower replication efficiency than ARS301 (Fig 2A, line 1) or ARS305 (Fig 2A, line 18).

A linker substitution mutation that disrupted the 11/11 ACS match inactivated ARS302 (Fig 2B, line 2). Similar mutations within each of the 10/11 matches did not greatly affect replication efficiency (lines 3–5). A mutation within the putative Abf1p binding site reduced replication efficiency by 50% (line 6). Mutations within the putative Rap1p binding site reduced replication efficiency even farther (Fig 3).

View larger version (51K): In this window In a new window Download PPT slide   Figure 3. Linker substitution analysis of ARS302. A 14-bp KpnI linker was used to scan the 275-bp ARS302 fragment in head-to-tail fashion, generating a total of 20 clones (ls20 has a 10-bp substitution). Additional substitutions were performed for the five regions covered by ls7, ls9, and ls11–ls13, utilizing a 7-bp KpnI linker. The results plotted are the averages of three independent experiments with standard deviations. In spite of repeated efforts, we were unable to clone ls13a in bacterial cells and therefore could not assess the effect of that substitution. We do not know why wild-type ARS302 replication efficiency was lower in this series of experiments (9%) than in the series reported in Fig 2B (20%), which was performed several months earlier. We note that within each series repetitions of the wild-type control were consistently high (Fig 2B) or low (Fig 3).

PCR-generated deletions from the 5' side of the essential ACS (Fig 2B, lines 7–9) all reduced ARS activity. Construct 1-39, which deletes the Abf1p binding site, reduced ARS activity to a level similar to that of the linker-substituted Abf1p site (Fig 2B, lines 6 and 7). Further deletions resulted in greater loss or total absence of ARS activity (Fig 2B, lines 8 and 9). Thus, C-domain sequences are required for ARS302 function even in the presence of an intact B domain. Progressive deletions within the B domain also compromised ARS activity (Fig 2B, lines 10–14).

Thus ARS302 contains essential A, B, and C domains. Although not individually essential, sequences at the extreme left and right ends of the 275-bp HincII-HindIII fragment contribute significantly to ARS function. For this reason we used the complete restriction fragment in subsequent experiments.

ARS302 is modular:
Because ARS302 is unusually large and has an unusually stringent requirement for C-domain sequences, we conducted higher resolution analysis of its sequence requirements by progressive linker substitution (ls). To accommodate the large size of ARS302, we initially used a large 14-bp KpnI linker (G + C rich) to generate 20 linker substitutions spanning the 275-bp fragment. Six substitutions (ls7 and ls9–13) completely abolished ARS activity (data not shown). To identify the important sequences within these regions at higher resolution, 7-bp KpnI linkers were employed. The 7-bp linker substitutions are designated ls7a, ls7b, etc. in Fig 3. Despite repeated efforts, we were unable to clone ls13a in bacterial cells. We did not subdivide ls10, which includes the essential ACS.

As in the case of linker substitutions targeting the ACS in other tested ARS elements (MARAHRENS and STILLMAN 1992 ; RAO et al. 1994 ; RASHID et al. 1994 ; THEIS and NEWLON 1994 ; HUANG and KOWALSKI 1996 ), ls10 abolished ARS function (Fig 3). But in contrast to other tested ARS elements, three additional linker substitutions also led to complete loss of ARS activity (Fig 3, ls9b, ls11b, and ls12a).

Consistent with Fig 2B, linker substitution revealed that sequences within the C domain contribute significantly to ARS302. ls3, which overlaps the putative Abf1p binding site, resulted in a small reduction in ARS activity, and ls6 and ls7a, which overlap the putative Rap1p binding site, had a greater effect (Fig 3). ls7b and ls9a also reduced ARS activity significantly, suggesting that the substituted sequences are important for ARS302 function.

Thus our mutational analysis of ARS302 revealed that it resembles chromosomally active nonsilencer-associated ARS elements in being modular, with an essential ACS and B domain. It differs from previously characterized ARS elements in having an essential C domain. Since ARS301 does not have an essential C domain (Fig 2A), it is unlikely that this feature of ARS302 is relevant to its chromosomal inactivity or its role in silencing. Rather, this feature is more likely to be another of the many possible variations in sequence and organization of S. cerevisiae ARS elements (RASHID et al. 1994 ; THEIS and NEWLON 1997 ).

The sequences flanking ARS301 can inactivate ARS305:
SANTOCANALE et al. 1999 , VUJCIC et al. 1999 , and WEINBERGER et al. 1999 previously demonstrated that ARS301 is inactive as an origin at its native chromosomal location because it is extremely late firing at this position. To test the role of flanking sequences in rendering ARS301 inactive, we replaced ARS301 with ARS305, which is an active, early-firing origin at its native chromosomal position (HUBERMAN et al. 1988 ; REYNOLDS et al. 1989 ; DUBEY et al. 1991 ; HUANG and KOWALSKI 1993 ; BOUSSET and DIFFLEY 1998 ). Specifically, we deleted the sequences corresponding to the 115-bp version of ARS301 (Fig 2A, line 16) and replaced them with a 550-bp NruI-ClaI restriction fragment containing ARS305 (HUANG and KOWALSKI 1993 ). The cartoon (not to scale) in Fig 4A shows the structure of the modified chromosome with an extra copy of ARS305 at the position of ARS301. Two-dimensional gel analysis of DNA from exponentially growing cells of this strain revealed that the extra copy of ARS305 generated a Y arc but not a bubble arc (Fig 5A and Fig B), indicating that in this new context ARS305 is passively replicated. A control experiment using the same DNA preparation (Fig 5C) revealed a strong bubble arc and weaker Y arc from ARS305 at its native position. Thus the absence of a bubble arc in Fig 5B could not be attributed to loss of bubble intermediates during DNA preparation. Since ARS301 at its normal location is inactive because it fires late in S phase (SANTOCANALE et al. 1999 ; VUJCIC et al. 1999 ; WEINBERGER et al. 1999 ), we assume that inactivation of ARS305 at this position is accomplished in a similar fashion—by forcing it to fire so late that it is passively replicated by the fork from the active copy of ARS305 before it can fire on its own.

View larger version (18K): In this window In a new window Download PPT slide   Figure 4. Cartoons summarizing ARS element transplacement experiments (not to scale). (A) Transplacement of a copy of ARS305 to the position normally occupied by ARS301. The experiment was designed to test whether ARS305 at the position of ARS301 would be active or inactive at its new location (question marks). (B) Transplacement of a copy of ARS301 to the position normally occupied by ARS305. The experiment was designed to test whether ARS301 at the position of ARS305 would be active or inactive at its new location (question marks).

View larger version (111K): In this window In a new window Download PPT slide   Figure 5. Two-dimensional gel evaluation of the activity of potential replication origins moved to new locations. (A) Diagram illustrating the positions of Y arcs (indicative of passive replication) and bubble arcs (indicative of active origin firing) after neutral-neutral two-dimensional gel electrophoresis (BREWER and FANGMAN 1987 ). (B and C) ARS305 is not active in the position of ARS301. A 550-bp NruI-ClaI fragment containing ARS305 was moved to the position of ARS301 (strain YKS108; Table 1). DNA was isolated from this strain and separated into two portions. The first portion was digested with EcoRV and BamHI and then subjected to 2-D gel electrophoresis. The blot was probed with a 1.2-kbp XbaI fragment covering ARS301 (B). This probe detects a 4.2-kbp EcoRV-BamHI fragment centered on the ectopic copy of ARS305. It also detects two smaller restriction fragments containing sequences similar to those flanking ARS301. These also generate Y arcs, which are partially visible in the right half of B. The second DNA portion was digested with EcoRV and HindIII and subjected to 2-D gel electrophoresis (C). The blot was probed with the 550-bp NruI-ClaI fragment containing ARS305. This probe detects a 4.7-kbp restriction fragment centered on ARS305 at its native location. Because the ectopic copy of ARS305 at the ARS301 position was inserted into that position as a HindIII cassette, it is excised by HindIII digestion as a small fragment that migrates out of the detectable range. (D–F) ARS301 is inactive at the position of ARS305. The 115-bp (D) and 95-bp (F) fragments containing ARS301 (Fig 2A, lines 16 and 17) were moved to the position of ARS305 (strains YKS100 and YKS101; Table 1). DNA was digested with NcoI and then subjected to 2-D gel electrophoresis. A 2.1-kbp BamHI-PstI fragment covering ARS305 was used to detect a 4.6-kbp NcoI restriction fragment centered on the transplaced copy of ARS301. In E, DNA from strain YKS100 was probed with a 1.4-kbp EcoRI fragment containing ARS1. This probe detects a 4.7-kbp NcoI fragment centered on ARS1. (G–I) ARS302 is not activated in the position of ARS305 or ARS1. The 275-bp HincII-HindIII fragment containing ARS302 was moved to the locations of ARS305 and ARS1 (strains YKS102 and YKS103; Table 1). Isolated DNA was cut with the indicated restriction enzymes and subjected to 2-D gel electrophoresis. A 4.6-kbp EcoRI fragment centered on the copy of ARS302 at the ARS305 position was detected by probing with a 2.1-kbp BamHI-PstI fragment covering ARS305 (G). A 4.7-kbp NcoI fragment centered on ARS302 at the ARS1 position was detected by probing with a 1.4-kbp EcoRI fragment covering ARS1 (H). The same blot was reprobed with the 2.1-kbp BamHI-PstI fragment covering ARS305 to reveal the 5.1-kbp NcoI fragment centered on ARS305 (I).

ARS301 and ARS302 are not activated when moved to positions occupied by active origins:
The above results suggest that the sequences flanking ARS301 are capable, on their own, of forcing an otherwise early-firing ARS element to fire late in S phase. We suspected, however, that sequences within ARS301 and ARS302 might also contribute to determination of late firing. To test this possibility, we moved ARS301 and ARS302 to other locations normally occupied by active replication origins. We chose the location of ARS305 on chromosome III (as diagrammed in Fig 4B) and the location of ARS1 on chromosome IV as recipient sites for transposition, since both ARS305 and ARS1 are well-characterized, early-firing, chromosomally active origins (MARAHRENS and STILLMAN 1992 ; HUANG and KOWALSKI 1993 , HUANG and KOWALSKI 1996 ; LIN and KOWALSKI 1997 ). In addition, the ARS1 location offered the opportunity to test whether origin inactivation is unique to chromosome III or also applies to other chromosomes.

Two-dimensional gel analyses (Fig 5, D–I) revealed that neither ARS301 nor ARS302 is significantly activated by transposition to the new locations. Constructs of 115 bp and 95 bp containing ARS301 (with or without its Rap1p binding site) at the ARS305 location display primarily Y arc signals, indicating that ARS301 at this position is passively replicated (presumably by the fork from ARS306; Fig 1 and Fig 4B) most of the time (Fig 5D and Fig F). Restriction fragments containing ARS302 at the ARS305 position (Fig 5G) or at the ARS1 position (Fig 5H) were also primarily passively replicated. In all cases, control experiments demonstrated the presence of bubble arcs at known active origins in the DNA preparations (Fig 5E and Fig I, and data not shown).

Thus, these results suggest that sequences within ARS301 and ARS302 are sufficient to prevent them from firing even after they have been moved to chromosomal locations usually occupied by active early-firing origins. Presumably these internal sequences dictate late origin firing. The fact that ARS302 is inactive at the position of ARS1 on chromosome IV demonstrates that the phenomenon of ARS element inactivation is not restricted to chromosome III.

ARS305 is not activated at the position of ARS301 in rad53 mutant cells:
SHIRAHIGE et al. 1998 demonstrated that a minimally active ARS element on chromosome VI, ARS608, could be further activated by mutation of the checkpoint gene, RAD53. This activation was due to accelerated firing of ARS608 in the mutant strain. In contrast, the results of SANTOCANALE et al. 1999 showed that ARS301 at its native location could not be activated solely by a rad53 mutation. The inability of ARS301 to function in the rad53 mutant strain is presumably a consequence of the combined effects of late-firing determinants within ARS301 itself and in its flanking sequences. Since ARS305 fires early at its native location, it is likely that ARS305 does not contain internal sequences specifying late firing. We therefore asked whether a copy of ARS305 moved to the position of ARS301—which is presumably rendered late firing only by flanking sequences—could be activated by a rad53 mutation.

We moved the 550-bp fragment containing ARS305 (the same fragment used in Fig 5B) to the position of ARS301 in rad53 mutant cells. Like the RAD53 strain (Fig 5B), the rad53 mutant strain generated only a Y arc (Fig 6A). When a 2-D gel of replication intermediates from the same DNA preparation was probed for ARS305 at its normal position, the usual bubble arc characteristic of ARS305 origin function was detected (Fig 6B). Thus the absence of a bubble arc in Fig 6A was not due to a defective DNA preparation. Instead, the presence of a Y arc without a bubble arc suggests that, even in a rad53 mutant strain, the sequences flanking ARS301 render ARS305 at the position of ARS301 sufficiently late firing that it cannot fire on its own before being passively replicated by the fork from ARS305 at its native position, where it is early firing.

View larger version (75K): In this window In a new window Download PPT slide   Figure 6. Effects of checkpoint mutations on activities of transplaced origins. (A) ARS305 at the position of ARS301 is not activated by rad53 mutation. The 550-bp fragment containing ARS305 activity was moved to the ARS301 location in rad53 mutant cells (strain YKS109; Table 1). The restriction enzymes and probe used were the same as in Fig 5B. (B) ARS305 is active at its native location in rad53 mutant cells. The same DNA preparation employed in A was digested with EcoRV-HindIII and was probed as in Fig 5C. (C and D) rad53 and mec1 mutations result in partial activation of ARS301 at the position of ARS305. The 115-bp fragment containing ARS301 (Fig 2A, line 16) was moved to the position of ARS305 in rad53 or mec1 mutant cells (strains YKS105 and YKS107; Table 1). The restriction fragments and probe utilized for the 2-D gel analysis are as in Fig 5D and Fig F. (E and F) ARS301 is inactive at the ARS305 position in the wild-type strains from which the rad53 and mec1 mutants used in C and D were derived. These analyses employed DNA from strains YKS104 (E) and YKS106 (F). Restriction fragments and probes were as in C and D.

ARS301 at the location of ARS305 is activated in rad53 and mec1 mutant cells:
We next wanted to check the effects of rad53 and mec1 mutations on the late firing determined by sequences internal to ARS301. For that purpose, we moved ARS301 (115-bp construct; Fig 2A, line 16) to the position of ARS305 in cells carrying mutant rad53 and mec1 genes. ARS301 became partially active in both the rad53 and mec1 mutant strains (Fig 6C and Fig D). However, when ARS301 was moved to the position of ARS305 in the corresponding wild-type strains, it remained inactive (Fig 6E and Fig F), as in strain YKS100 (Fig 5D). Thus, in the rad53 and mec1 mutant strains, ARS301 at the ARS305 location is rendered sufficiently early firing that in some cells it can fire before being inactivated by the fork arriving from early-firing ARS306.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Until this study, the chromosomal locations of ARS301 and ARS302 had been determined only approximately (BROACH et al. 1982 ). Nevertheless, on the basis of this approximate localization, it was possible to conclude that ARS301 and ARS302 are associated with the HML-E and -I silencers and are normally inactive as chromosomal replication origins (Fig 1; DUBEY et al. 1991 ). Recently, it was demonstrated (SANTOCANALE et al. 1999 ; VUJCIC et al. 1999 ; WEINBERGER et al. 1999 ) that the inactivity of ARS301 and probably ARS302 is due to these ARS elements being programmed to fire extremely late in S phase. The experiments described here were designed to more precisely determine the locations of ARS301 and ARS302, to test whether their sequence organizations differ significantly from those of active nonsilencer-associated origins, and to evaluate the contributions of both internal and flanking sequences to their inactivity as chromosomal origins.

ARS301 and ARS302 resemble other ARS elements:
Our analysis of ARS301 (Fig 2A) revealed that a stretch of 95 bp (the essential ACS match, 10 bp 5' of the ACS and 74 bp 3' of the ACS) is sufficient for ARS activity. In contrast to our results for ARS301 (Fig 2A) and to results from other labs for other ARS elements (reviewed in THEIS and NEWLON 1996 ), our analysis of ARS302 revealed that sequences 5' of the essential ACS match (C domain) as well as sequences 3' of the ACS (B domain) are absolutely required for activity. For this reason, ARS302 is larger than the typical S. cerevisiae ARS element.

Systematic linker substitutions of ARS302 (Fig 3) revealed that it has a modular structure similar to those of other ARS elements (MARAHRENS and STILLMAN 1992 ; RAO et al. 1994 ; RASHID et al. 1994 ; THEIS and NEWLON 1994 ; HUANG and KOWALSKI 1996 ). The A domain of ARS302 (ls9b and ls10) is essential, and so are the sequences that correspond in position to the B1 component of other ARS elements (ls11b and ls12a). Additional B-domain sequences (ls13 and ls14) that correspond in position to the B2 element of ARS1 and ARS307 are also important. Important C-domain sequences include ls9a, which is adjacent to the A domain. Additional experiments are needed to determine whether ls9a inhibits the ORC binding function of the adjacent A domain. It seems likely that ls6 and ls7a in the C domain inhibit ARS302 by interfering with Rap1p binding. Similarly, the mild inhibition by ls3 may be due to interference with Abf1p binding. The causes of the inhibitions by ls1, ls5, and ls7b are not clear. Thus, although ARS302 differs from other ARS elements in some respects, both ARS301 and ARS302 resemble them in others, especially with regard to sequences required for ORC binding. Consistent with this observation is the demonstration that ORC binds to ARS301 and ARS302 in vitro (BELL et al. 1993 ), and a prereplicative complex containing ORC and additional proteins is assembled at ARS301 in vivo (SANTOCANALE and DIFFLEY 1996 ).

Although our survey of the sequence requirements for plasmid origin function by ARS301 and ARS302 did not reveal unusual structural features of these ARS elements (Fig 2 and Fig 3), the survey did emphasize that both ARS elements are located close to binding sites for transcription factors (Abf1p and Rap1p) that have been implicated in transcriptional silencing (BRAND et al. 1987 ; MAHONEY and BROACH 1989 ; MAHONEY et al. 1991 ). It seems likely that it is the spacing and density of ORC, Rap1p, and Abf1p binding sites that distinguishes nonsilencer-associated from silencer-associated origins. The results presented here indicate that the Rap1p and Abf1p binding sites close to silencer-associated origins may (as in the case of the Rap1p site in ARS302; Fig 3) or may not (as in the case of the Rap1p site near ARS301; Fig 2) contribute significantly to plasmid replication origin activity.

Our results and those from earlier studies (NEWLON et al. 1991 , NEWLON et al. 1993 ) indicate that only one origin, ARS301, is closely associated with the HML-E silencer. In contrast, the HML-I silencer is closely associated with three origins: ARS302, which is part of HML-I, and ARS303 and ARS320, which are <2 kbp away (Fig 1). ARS302, ARS303, and ARS320 are all individually weak ARS elements. Several individually weak origins are similarly clustered close to the HMR-E silencer near the right end of chromosome III (DEBEER and FOX 1999 ; HURST and RIVIER 1999 ). It is possible that interactions between closely spaced weak origins may contribute to silencer function in some cases (DEBEER and FOX 1999 ; HURST and RIVIER 1999 ).

In addition to the evidence provided here, further evidence that ability to function as a replication origin in a plasmid does not correlate with ability to serve as a replication origin in chromosomes comes from the following observations: (1) ARS301 and ARS305 are both efficient replication origins in plasmids (Fig 2A, lines 1 and 18), yet ARS305 is a chromosomal origin and ARS301 is not; and (2) ARS302 and ARS307 are both inefficient plasmid origins (Fig 2B and Fig 3; THEIS and NEWLON 1994 ), yet ARS307 is a chromosomal origin and ARS302 is not. Taken together, the results summarized above suggest that other factors—not ARS structure and not plasmid origin efficiency—dictate whether or not an origin can fire in the chromosome.

Flanking sequences contribute to maintaining ARS301 inactive:
SANTOCANALE et al. 1999 , VUJCIC et al. 1999 , and WEINBERGER et al. 1999 have recently shown that these other factors involve control of replication timing. ARS301 and ARS302 are rendered inactive because they are programmed to fire extremely late during S phase. For this reason, they are always passively replicated by the fork from early-firing ARS305 before they have a chance to fire on their own.

To address the contribution of sequences flanking ARS301 to rendering it inactive, we moved ARS305, which normally fires very early (REYNOLDS et al. 1989 ; BOUSSET and DIFFLEY 1998 ), to the position of ARS301 (Fig 4A). We found that ARS305 was inactivated at its new location (Fig 5B). Even in rad53 mutant cells, ARS305 at the position of ARS301 was inactive (Fig 6A). The simplest explanation of these observations is that sequences flanking ARS301 are capable of rendering any origin inserted at the position of ARS301 so late firing that, before it can fire on its own, it is passively replicated by the fork from ARS305. This effect is not blocked even when the firing of late origins is accelerated due to rad53 mutation. Since the sequences flanking ARS301 influence the activity of ARS305 when it is substituted for ARS301, it is reasonable to infer that these sequences contribute to maintaining ARS301 inactive in the chromosome.

ARS301 and ARS302 may contain internal late-firing determinants:
Earlier experiments suggested that the default time for origin firing is early and that ARS elements within certain regions of the S. cerevisiae genome fire later than others due to the influence of flanking sequences (FERGUSON and FANGMAN 1992 ; FRIEDMAN et al. 1997 ). That this might not be the case for ARS301 was suggested by BOUSSET and DIFFLEY'S 1998 observation that a plasmid (pCS1) containing ARS301 with short 5' and 3' flanks replicates in late S phase. Our measurements of ARS301 location and size (Fig 2A) now permit the conclusion that the 208-bp insert in late-replicating pCS1 includes the 95-bp minimal ARS301 (Fig 2A, line 17), 93 bp of 5' flanking sequence, and 20 bp of 3' flanking sequence. These measurements raise the possibility that the determinants of late origin firing in pCS1 may lie (at least in part) within the minimal ARS element rather than within the short flanking sequences.

To assess the role in replication timing and origin activity of sequences within minimal ARS301 and ARS302, we moved minimal versions of both ARS elements to the position of ARS305 (Fig 4B) and asked if either was now activated. In addition, we also transposed ARS302 to the position of ARS1 on chromosome IV to address the possible role of specific chromosomal context in its inactivity. The results in Fig 5 show that, even at positions normally occupied by active origins, minimal ARS301 and ARS302 are unable to significantly initiate replication. This suggests that, even though they have now been separated from their normal flanking sequences, their time of firing at the ectopic locations is not sufficiently advanced to allow them to initiate replication before being passively replicated by forks from nearby early-firing origins. Since both ARS305 and ARS1 are active early-firing replication origins at their normal locations, it is unlikely that the sequences flanking them dictate late origin firing. Thus, the internal sequences within minimal ARS301 and ARS302 appear to be capable of specifying late origin firing on their own. This capability is not confined to chromosome III, as ARS302 is also inactive at the position of ARS1 on chromosome IV.

To address the regulation of late firing specified by the internal sequences, we asked if ARS301 could be activated when moved to the ARS305 position in mec1 and rad53 checkpoint-deficient cells. We observed that ARS301 at the ARS305 location became partially active in rad53 and mec1 mutant strains (Fig 6C and Fig D). This observation suggests that ARS301 is prevented from firing at the ARS305 position by an active checkpoint mechanism dependent on the Mec1 and Rad53 proteins and this mechanism acts at least in part on the minimal ARS301 sequence that was transposed to the ARS305 position. Presumably this is the same Mec1p- and Rad53p-dependent mechanism that was previously demonstrated to retard the activation of late-firing origins (SANTOCANALE and DIFFLEY 1998 ; SHIRAHIGE et al. 1998 ).

These results imply that ARS301 is inherently late firing, and some of the sequences responsible for that late firing are located within a stretch of 95 bp. This provides an excellent starting point for the precise localization of cis-acting sequences responsible for determining the time of firing of a eukaryotic origin.

Both internal and flanking sequences contribute to rendering ARS301 and ARS302 inactive in the chromosome:
Thus our results suggest that in their normal chromosomal positions ARS301 and ARS302 are inactivated by two effects, each of which contributes to late firing. Late firing is imposed first by internal sequences within the ARS element itself, which prevent minimal ARS301 and ARS302 from being activated at the ARS305 position, and second by flanking sequences, which are capable of rendering even a normally early-firing origin (e.g., ARS305) late firing. It is not surprising, therefore, that activation of ARS301 and ARS302 in their normal chromosomal positions requires draconian measures (SANTOCANALE et al. 1999 ; VUJCIC et al. 1999 ; WEINBERGER et al. 1999 ).

	ACKNOWLEDGMENTS

We thank Rolf Sternglanz, John Diffley, and Deborah Mahoney for providing yeast strains; and we thank the anonymous referees for helpful suggestions. This research was supported by grants R01-GM49294 and R21-CA84302 from the National Institutes of Health and by a 1999 Developmental Funds award from the Roswell Park Cancer Center Support Grant (P30 CA16056-24).

Manuscript received July 6, 2000; Accepted for publication June 1, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALLEN, J., Z. ZHOU, W. SIEDE, E. FRIEDBERG, and S. ELLEDGE, 1994  The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev. 8:2401-2415[Abstract/Free Full Text].

BELL, S. P., R. KOBAYASHI, and B. STILLMAN, 1993  Yeast origin recognition complex functions in transcription silencing and DNA replication. Science 262:1844-1849[Abstract/Free Full Text].

BELL, S. P. and B. STILLMAN, 1992  ATP dependent recognition of eukaryotic origins of DNA replication by a multi-protein complex. Nature 357:128-134[Medline].

BOEKE, J. D., J. TRUEHEART, G. NATSOULIS, and G. R. FINK, 1987  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175[Medline].

BOUSSET, K. and J. F. X. DIFFLEY, 1998  The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev. 12:480-490[Abstract/Free Full Text].

BRAND, A. H., G. MICKLEM, and K. NASMYTH, 1987  A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell 51:709-719[Medline].

BREWER, B. J. and W. L. FANGMAN, 1987  The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463-471[Medline].

BROACH, J. R., Y.-Y. LI, J. FELDMAN, M. JAYARAM, and J. ABRAHAM et al., 1982  Localization and sequence analysis of yeast origins of DNA replication. Cold Spring Harbor Symp. Quant. Biol. 47:1165-1173.

BUCHMAN, A. R., W. J. KIMMERLY, J. RINE, and R. D. KORNBERG, 1988  Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol. Cell. Biol. 8:210-225[Abstract/Free Full Text].

DEBEER, M. P. and C. A. FOX, 1999  A role for a replicator dominance mechanism in silencing. EMBO J. 18:3808-3819[Medline].

DIFFLEY, J. F. X. and J. H. COCKER, 1992  Protein-DNA interactions at a yeast replication origin. Nature 357:169-172[Medline].

DUBEY, D. D., L. R. DAVIS, S. A. GREENFEDER, L. Y. ONG, and J. ZHU et al., 1991  Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus do not function as chromosomal DNA replication origins. Mol. Cell. Biol. 11:5346-5355[Abstract/Free Full Text].

FERGUSON, B. M. and W. L. FANGMAN, 1992  A position effect on the time of replication origin activation in yeast. Cell 68:333-339[Medline].

FRIEDMAN, K. L., B. J. BREWER, and W. L. FANGMAN, 1997  Replication profile of Saccharomyces cerevisiae chromosome VI. Genes Cells 2:667-678[Abstract].

HUANG, R.-Y. and D. KOWALSKI, 1993  A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome. EMBO J. 12:4521-4531[Medline].

HUANG, R.-Y. and D. KOWALSKI, 1996  Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome. Nucleic Acids Res. 24:816-823[Abstract/Free Full Text].

HUBERMAN, J. A., L. D. SPOTILA, K. A. NOWOTKA, S. M. EL-ASSOULI, and L. R. DAVIS, 1987  The in vivo replication origin of the yeast 2 micron plasmid. Cell 51:473-481[Medline].

HUBERMAN, J. A., J. ZHU, L. R. DAVIS, and C. S. NEWLON, 1988  Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 16:6373-6384[Abstract/Free Full Text].

HURST, S. T. and D. H. RIVIER, 1999  Identification of a compound origin of replication at the HMR-E locus in Saccharomyces cerevisiae. J. Biol. Chem. 274:4155-4159[Abstract/Free Full Text].

LAURENSON, P. and J. RINE, 1992  Silencers, silencing, and heritable transcriptional states. Microbiol. Rev. 56:543-560[Abstract/Free Full Text].

LIN, S. and D. KOWALSKI, 1997  Functional equivalency and diversity of cis-acting elements among yeast replication origins. Mol. Cell. Biol. 17:5473-5484[Abstract].

LUSTIG, A. J., 1998  Mechanisms of silencing in Saccharomyces cerevisiae. Curr. Opin. Genet. Dev. 8:233-239[Medline].

MAHONEY, D. J. and J. R. BROACH, 1989  The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers. Mol. Cell. Biol. 9:4621-4630[Abstract/Free Full Text].

MAHONEY, D. J., R. MARQUARDT, G.-J. SHEI, A. B. ROSE, and J. R. BROACH, 1991  Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression. Genes Dev. 5:605-615[Abstract/Free Full Text].

MARAHRENS, Y. and B. STILLMAN, 1992  A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255:817-823[Abstract/Free Full Text].

NEWLON, C. S., L. R. LIPCHITZ, I. COLLINS, A. DESHPANDE, and R. J. DEVENISH et al., 1991  Analysis of a circular derivative of Saccharomyces cerevisiae chromosome III: a physical map and identification and location of ARS elements. Genetics 129:343-357[Abstract].

NEWLON, C. S., I. COLLINS, A. DERSHOWITZ, A. M. DESHPANDE, and S. A. GREENFEDER et al., 1993  Analysis of replication origin function on chromosome III of Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 58:415-423[Medline].

PAULOVICH, A. G., R. U. MARGULIES, B. M. GARVIK, and L. H. HARTWELL, 1997  RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage. Genetics 145:45-62[Abstract].

RAO, H., Y. MARAHRENS, and B. STILLMAN, 1994  Functional conservation of multiple elements in yeast chromosomal replicators. Mol. Cell. Biol. 14:7643-7651[Abstract/Free Full Text].

RAO, H. and B. STILLMAN, 1995  The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc. Natl. Acad. Sci. USA 92:2224-2228[Abstract/Free Full Text].

RASHID, M. B., K. SHIRAHIGE, N. OGASAWARA, and H. YOSHIKAWA, 1994  Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae. Gene 150:213-220[Medline].

REYNOLDS, A. E., R. M. MCCARROLL, C. S. NEWLON, and W. L. FANGMAN, 1989  Time of replication of ARS elements along yeast chromosome III. Mol. Cell. Biol. 9:4488-4494[Abstract/Free Full Text].

RIVIER, D. H., J. L. EKENA, and J. RINE, 1999  HMR-I is an origin of replication and a silencer in Saccharomyces cerevisiae. Genetics 151:521-529[Abstract/Free Full Text].

RIVIER, D. H. and J. RINE, 1992  An origin of DNA replication and a transcription silencer require a common element. Science 256:659-663[Abstract/Free Full Text].

ROWLEY, A., J. H. COCKER, J. HARWOOD, and J. F. X. DIFFLEY, 1995  Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC. EMBO J. 14:2631-2641[Medline].

SAAVEDRA, R. A. and J. A. HUBERMAN, 1986  Both DNA topoisomerases I and II relax 2µm plasmid DNA in living yeast cells. Cell 45:65-70[Medline].

SANTOCANALE, C. and J. F. X. DIFFLEY, 1996  ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J. 15:6671-6679[Medline].

SANTOCANALE, C. and J. F. X. DIFFLEY, 1998  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395:615-618[Medline].

SANTOCANALE, C., K. SHARMA, and J. F. X. DIFFLEY, 1999  Activation of dormant origins of DNA replication in budding yeast. Genes Dev. 13:2360-2364[Abstract/Free Full Text].

SCHERER, S. and R. W. DAVIS, 1979  Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. USA 76:4951-4955[Abstract/Free Full Text].

SHARMA, K., 2000 Characterization and functional analysis of HML-silencer-associated ARS elements in Saccharomyces cerevisiae. Ph.D. Thesis, State University of New York, Buffalo, NY.

SHERMAN, F., 1991  Getting started with yeast. Methods Enzymol. 194:3-21[Medline].

SHIRAHIGE, K., Y. HORI, K. SHIRASHI, M. YAMASHITA, and K. TAKAHASHI et al., 1998  Regulation of DNA-replication origins during cell-cycle progression. Nature 395:618-621[Medline].

SIKORSKI, R. S. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27[Abstract/Free Full Text].

STONE, E. M. and L. PILLUS, 1998  Silent chromatin in yeast: an orchestrated medley featuring Sir3p. Bioessays 20:30-40[Medline].

THEIS, J. F. and C. S. NEWLON, 1994  Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function. Mol. Cell. Biol. 14:7652-7659[Abstract/Free Full Text].

THEIS, J. F., and C. S. NEWLON, 1996 The replication of yeast chromosomes, pp. 3–28 in The Mycota III: Biochemistry and Molecular Biology, edited by R. BRAMBL and G. A. MARZLUF. Springer-Verlag, Berlin.

THEIS, J. F. and C. S. NEWLON, 1997  The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence. Proc. Natl. Acad. Sci. USA 94:10786-10791[Abstract/Free Full Text].

VUJCIC, M., C. A. MILLER, and D. KOWALSKI, 1999  Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast. Mol. Cell. Biol. 19:6098-6109[Abstract/Free Full Text].

WALKER, S. S., S. C. FRANCESCONI, and S. EISENBERG, 1990  A DNA replication enhancer in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 87:4665-4669[Abstract/Free Full Text].

WEINBERGER, M., P. A. TRABOLD, M. LU, K. SHARMA, and J. A. HUBERMAN et al., 1999  Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae. J. Biol. Chem. 274:35975-35984[Abstract/Free Full Text].

ZHU, J., C. S. NEWLON, and J. A. HUBERMAN, 1992  Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae. Mol. Cell. Biol. 12:4733-4741[Abstract/Free Full Text].

This article has been cited by other articles:

				L. Casey, E. E. Patterson, U. Muller, and C. A. Fox Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S Phase Cyclin Mol. Biol. Cell, February 1, 2008; 19(2): 608 - 622. [Abstract] [Full Text] [PDF]