The Roles of Klenow Processing and Flap Processing Activities of DNA Polymerase I in Chromosome Instability in Escherichia coli K12 Strains

Corresponding author: Kazuo Yamamoto, Graduate School of Life Sciences, Tohoku University, Sendai 9808578, Japan., yamamot{at}mail.cc.tohoku.ac.jp (E-mail)

Communicating editor: R. MAURER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the polA and polA107 mutant strains were compared. Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations. The polA strain, which is deficient in both Klenow domain and 5' 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1–4. The polA107 strain, which is deficient in the 5' 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2. Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4. The 5' 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases. We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA.

DNA polymerase I (PolI) of Escherichia coli has three enzymatic activities acting as a 5' 3' DNA polymerase, a 3' 5' exonuclease that edits 3' terminal nucleotides of nascent DNA, and a 5' 3' exonuclease that removes nucleotides from the 5' end of DNA or RNA (KORNBERG and BAKER 1992 ). Proteolytic cleavage of PolI separates the polypeptide chain into two active fragments (BRUTLAG et al. 1969 ): the large C-terminal fragment called "Klenow" contains the 5' 3' polymerase and 3' 5' exonuclease activities, and the small N-terminal contains only 5' 3' exonuclease activity. The 5' 3' exonuclease is a homolog of mammalian FEN1 and yeast RAD27 flap endonucleases (ROBINS et al. 1994 ).

PolI is believed to be involved in both DNA replication and repair (see KORNBERG and BAKER 1992 ). During replication, PolI is thought to remove the RNA primer that initiates Okazaki fragments replacing short oligonucleotides with DNA. During repair, PolI can bind to single-strand breaks in the phosphodiester backbone, excise the damaged region, and restore the integrity of the double helix.

Mutations in the polA gene have been shown to influence the frequencies of chromosomal deletion and minus frameshift mutations (COUKELL and YANOFSKY 1970 ; VACCARO and SIEGEL 1975 ; SAVIC and ROMAC 1982 ; FIX et al. 1987 ). We investigated spontaneous mutagenesis in the endogenous tonB gene of the E. coli polA1 strain and found marked increases in deletions and frameshifts (AGEMIZU et al. 1999 ). The polA1 mutation provides normal 5' 3' exonuclease activity with no Klenow activity (JOYCE et al. 1985 ). Inspection of the sequences around deletion end-points and frameshift points indicated that these mutations occurred at G:C base pair clusters; for example, among the 20 deletion sites recovered, ranging in sizes from 4 to 12,532 bp, 8 sites were associated with the GC clusters at both ends of the junctions and 10 at either end of the junction, and among 6 minus frameshifts, 5 were associated with a GC cluster. Thus, there seems to be a resemblance between deletions and frameshifts recovered from the polA1 strain and the phenomenon known as instability of microsatellite repetitive sequences.

Length variation of simple repetitive DNA sequences is known to be associated with human colon cancer (AALTONEN et al. 1993 ; IONOV et al. 1993 ; THIBODEAU et al. 1993 ). Human cells that exhibit microsatellite instability are frequently deficient in mismatch repair (KROUTIL et al. 1996 ). A substantial fraction of familial colorectal cancer is associated with microsatellite instability and mutations in mismatch repair genes (KOLODNER 1995 ). Defects in DNA mismatch repair in E. coli and yeast result in an increase in the instability of repetitive sequences (LEVINSON and GUTMAN 1987 ; STRAND et al. 1993 ). Thus, the mismatch repair system can stabilize microsatellites. However, about half of the sporadic tumor cell lines with microsatellite instability do not contain mutations in the mismatch repair genes, which suggests that other genes may also contribute to microsatellite instability (LIU et al. 1995 ). Mutations in the RAD27 Saccharomyces cerevisiae gene, a homolog of 5' 3' exonuclease of PolI, also destabilize microsatellites by a mechanism distinct from mismatch repair (JOHNSON et al. 1995 ; TISHKOFF et al. 1997 ; KOKOSKA et al. 1998 ). A defect in the 5' 3' exonuclease of PolI has been shown to lead to an increase in poly(AC) repeat tract expansion (MOREL et al. 1998 ). These results strongly suggest that PolI may contribute to prevent the instability of repetitive sequences as one of the factors other than the mismatch repair system in E. coli.

In this study, we examined the sequence specificity of spontaneous mutations in the endogenous tonB gene of strains deficient in PolI activities. As PolI-deficient strains, we chose polA and polA107 alleles, deficient in both Klenow and 5' 3' exonuclease domains and 5' 3' exonuclease, respectively. Study of spontaneous mutations in the polA strain suggested an enhanced frequency of deletion, minus frameshift, plus frameshift, and duplication at GC cluster sites and in the polA107 strain revealed an enhanced frequency of plus frameshift and duplication at GC cluster sites.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains:
E. coli K12 strains KK102 (polA::Km^r zig219::Tn10) and KK103 (polA107 zig219::Tn10), which are a derivative of polA⁺ strain KK1 (WANG et al. 1996 ), were used for collecting mutations. KK1 carries the Cm^r gene (cat), located 1.6 kb upstream of the tonB gene on the chromosome (KITAMURA et al. 1995 ). The polA::Km^r allele, polA107 allele, and zig219::Tn10 allele were derived from strains CJ278 (polA::Km^r; JOYCE and GRINDLEY 1984 ), KL406 (polA107; National Institute of Genetics, Japan), and ET1214 (zig219:: Tn10; PAHEL et al. 1979 ), respectively. To construct isogenic polA strains, the zig219::Tn10 allele was first transduced into CJ278 or KL406 using P1 phage (IHARA et al. 1985 ) and UV-sensitive (UV^s) and tetracycline-resistant (Tet^r) derivatives were screened. KK1 was infected with P1 phage grown in resultant strains and selected for Tet^r transductants that were screened for UV^s. Colicinogenic E. coli strain CA18 carries colicin B factor (ISHII and KONDO 1972 ). T1 bacteriophage were included in the colicin plates.

Reagents and media:
Luria broth, L agar, and phosphate buffer were prepared as described by AKASAKA and YAMAMOTO 1991 . Colicin plates were prepared as described by UEMATSU et al. 1999 . Chloramphenicol (Cm; 30 µg/ml), ampicillin (50 µg/ml), or kanamycin (Km; 50 µg/ml) were included, if necessary, in Luria broth or L agar. Enzymes and reagents used for DNA manipulation were purchased from Takara Shuzo (Kyoto, Japan) and Applied Biosystems (Foster City, CA).

Mutation rate:
Mutation to colicin B and T1 phage resistance (ColB^r) was determined for 10 independent cultures of KK1 (polA⁺), KK101 (polA1), KK102 (polA), and KK103 (polA107) in 3 ml of Luria broth after overnight growth. For assays scoring ColB^r, independent cultures were plated directly onto colicin plate. ColB^r colonies were scored after 48 hr incubation at 37°. Total viable cells were determined by serial dilution with phosphate buffer, followed by plating on L agar. Mutation rates, expressed as mutations per cell per generation, were calculated by the method of the median (LEA and COULSON 1949 ) using the following formula: mutation rate = M/N, where M is the calculated number of mutation events and N is the mean number of viable cells in the culture. M is solved by interpolation from experimental determination of r_o, the median number of ColB^r cells determined among the cultures by the formula r_o = M(1.24 + ln M). Analysis of variance (ANOVA) was used to examine differences in mutation rate among the strains polA⁺, polA1, polA, and polA107.

Sequencing of the mutant DNA:
For the tonB mutation assay, independent colonies of KK102 (polA::Km^r) or KK103 (polA107) were inoculated in 3 ml of Luria broth at 37° overnight, and 100-µl aliquots of these overnight cultures were plated on colicin plates to obtain several hundred mutant colonies per plate. To collect tonB mutants, only one colB^r colony was chosen from each colicin plate, an approach that ensured each mutant analyzed was of independent origin. The DNA fragment including the mutant tonB gene was amplified by polymerase chain reaction (PCR) using appropriate primers (Fig 1) from genomic DNA that had been extracted from the tonB mutant. After amplification, the concentration of the amplified DNA was determined from the intensity of the band of the proper size on electrophoresis of 1-µl samples on 0.7% agarose gels. Mutant sequences were determined by the dideoxy chain termination method using an ABI automated sequencer model 373A.

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. Physical map of min 28 of E. coli linkage map tonB-trp region and the locations of primers for PCR amplification and sequencing (YAMAMURA et al. 2000 ). Relevant restriction sites with nucleotide numbers of the 19,495 bp DNA sequences in which 1 is the first G of the GATC BamHI site are included. In this numbering, cat is from base pair 377–1036, tonB from base pair 2993–3727, and trp from base pair 14,864–8334. Arrows below the map indicate the primers used: A (5'-AAACGTGCTAAATGTGCCGG-3', base pair 2702–2721), B (5'-CGATGTGGTGTGCTGCTATGC-3', base pair 3953–3933), C (5'-GATCATCTTCAGGCAAGT-3', base pair 1351–1368), D (5'-GGATTAAAACCTTCGCTTAA-3', base pair 1689–1708), E (5'-ATGGAAAATATCAAGCAAAT-3', base pair 2039–2058), F (5'-ATTATCTGCTTGTGGTGGTG-3', base pair 2389–2404), G (5'-GGCTTCCATGTCGGCAGAAT-3', base pair 968–987), 1 (5'-TAAGGCCATGCATAAAGT-3', base pair 2868–2885), 2.5 (5'-AGAAGCACCGGTGGTCA-3', 3256–3272), O1 (5'-CCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTC-3, base pair 545–584), O2 (5'-CTGGCCCGCATCCTTATCCGACCATTGTGCGTGAGTTTC-3', base pair 9760–9722), I1 (5'-ATCCGGAATTCCGTATGGCAATGAAAGACGGTGAG-3', base pair 585–619), I2 (5'-AGCGGATGATTGGCGAAGAAACCAAAGCGCAGATT-3', base pair 9721–9687), U1 (5'-GCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGG-3', base pair 200–237), U2 (5'-AGGCTTGTCATCATCGCGGGCAAACAAAAGCTCAAGGG-3', base pair 18029–17992), I1 (5'-ATCCGGAATTCCGTATGGCAATGAAAGACGGTGAG-3', base pair 585–619), I2 (5'-AGCGGATGATTGGCGAAGAAACCAAAGCGCAGATT-3', base pair 9721–9687).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To extend our previous study of the effects of polA1 mutation on spontaneous formation of tonB mutants (AGEMIZU et al. 1999 ), we measured the efficiency of spontaneous tonB mutations of KK102 (polA) and KK103 (polA107). In these cases, the polA1 mutation eliminates most of the Klenow activity but does not affect 5' 3' exonuclease activity (JOYCE et al. 1985 ), whereas the polA107 mutant lacks 5' 3' exonuclease activity but retains almost normal Klenow activity (HEIJNEKER et al. 1993). The polA mutation is a null mutation of the polA gene (JOYCE and GRINDLEY 1984 ). The presence of polA mutations was verified by examining UV sensitivity.

We first measured the spontaneous mutation rate of KK1 (polA⁺), KK101 (polA1), KK102 (polA), and KK103 (polA107), which were selected as the colB^r phenotype. The ColB^r mutation rate of KK1 was 1.91 x 10^-8; of KK101, 2.29 x 10^-7; of KK102, 1.52 x 10^-7; and of KK103, 9.70 x 10^-8 (Table 1). ANOVA was used to examine differences in mutation rates among four strains and significant differences could be found among them (F3, 8 = 14.382 and P = 0.0002). Fisher's post-hoc test showed that mutation rates were significantly different between polA⁺ and polA1 (P = 0.002), polA⁺ and polA (P = 0.0038), and polA⁺ and polA107 (P = 0.046).

A total of 65 independent ColB^r mutated clones from KK102 and 67 from KK103 were collected and used for DNA sequencing. DNA sequence analysis yielded a mutant sequence in 51 and 51 of these clones of KK102 and KK103, respectively. Thus, we were not able to identify the tonB gene mutation from 14 ColB^r clones of KK102 and 16 ColB^r clones of KK103. For comparison, their distribution by class is listed in Table 2 along with previously published results from polA1 strain UA1 (AGEMIZU et al. 1999 ) and polA⁺ strain TM31 (KITAMURA et al. 1995 ); both UA1 and TM31 are not isogenic from KK102 and KK103. The most frequent mutational event in the polA strain was deletion followed by -1 frameshift, base substitution, and +1 frameshift in that order. The types of mutation detected in the polA strain were considerably different from those of the polA107 strain where +1 frameshift dominated followed by duplication, deletion, base substitution, and -1 frameshift in that order.

Deletion:
As shown in Table 1 and Table 2, the rates of deletions in the polA1 strain (22.89 x 10^-8 x 39/61 = 14.65 x 10^-8) and the polA strain (15.19 x 10^-8 x 21/51 = 6.23 x 10^-8) were 38.5- and 16.4-fold, respectively, higher than that of the polA⁺ strain (1.91 x 10^-8 x 10/51 = 0.38 x 10^-8). Table 3 shows the 21 deletions identified from the polA strain, among which 12 were different sites, ranging in size from 4 to 102 bp. Six of these sites were flanked by repeated sequences (Table 3, boldface letters) of two or more bases, implying a role of direct repeats for deletion formation (ALBERTINI et al. 1982 ). Four of the 12 deletion sites showed 2- to 4-bp repeats (Table 3, italic letters) in the immediate vicinity of the endpoints, which appeared to indicate no clear association with deletion. Formation of deletion endpoints between these homologies can be explained if it is assumed that one or two extra bases have been added or deleted (UEMATSU et al. 1999 ). Three deletion sites had no homology at the endpoint. Further analysis of the DNA sequences at the sites of deletion events of the polA strain revealed that six had a GC cluster (>4-bp repeats; Table 3) within 3 bp of both endpoints. In addition, five sites had a GC cluster within 3 bp of one endpoint. Furthermore, 7 of the 12 sites had imperfect inverted repeats at one or both ends of the endpoints (Table 3, arrow). Among 21 deletions, 1 particular deletion (13 bp, nucleotides 3023–3035) accounted for nine incidences. This site was also a very strong deletion hotspot site in the polA1 strain (17 among 39 deletions; AGEMIZU et al. 1999 ).

View this table: In this window In a new window   Table 3. Target sequences of deletion mutations in a polA or polA107 strain

Table 3 also shows seven deletions identified in the KK103 (polA107) strain. Spontaneous deletion rate of KK103 was 1.33 x 10^-8 (9.70 x 10^-8 x 7/51), which was not significantly different from that observed in the polA⁺ strain (² = 0.454, P = 0.499). All of the deletions were flanked by repeated sequences. In addition, all the deletions of the polA107 strain had a GC cluster at both of the deletion endpoints and had imperfect inverted repeats. Deletion at nucleotides 3395–3496 was counted three times and was also detected once in the polA strain. Deletion at nucleotides 3437–3474 was detected in both KK102 and KK103 strains.

Frameshift:
Table 1 and Table 2 show that the frameshift rate in KK101 (polA1), KK102 (polA), and KK103 (polA107) was 25.9-, 41.0-, and 36.3-fold higher than that in the polA⁺ strain, respectively. Previous studies of spontaneous mutagenesis in the polA1 strain (VACCARO and SIEGEL 1975 ; SAVIC and ROMAC 1982 ; FIX et al. 1987 ; AGEMIZU et al. 1999 ) suggested the presence of minus frameshifts. Table 2 shows that 9 of 16 frameshifts in the polA strain resulted from the loss of a base pair and 7 were due to a base pair gain. Two minus frameshifts among 22 recovered in the present study of the polA107 strain were observed and 20 plus frameshifts were also observed. Thus, the polA107 strain is a plus frameshift mutator and the polA strain is a plus/minus frameshift mutator. On the other hand, the polA1 strain is a minus frameshift mutator.

The DNA sequences surrounding each of the minus and plus frameshift mutations recovered are depicted in Table 4 and Table 5, respectively. Table 4 indicates that four of nine cases of minus frameshifts recovered from the polA strain could be explained on the basis of slippage events in runs of direct sequences (STREISINGER et al. 1966 ). On the other hand, the remaining five cases of frameshift mutation in the polA strain and two cases in the polA107 strain occurred at nonruns (Table 4). Eight of 9 minus frameshifts in the polA strain were associated with GC clusters (Table 4). All the minus frameshifts, except 3082–3084, were also associated with imperfect inverted repeats. Table 5 illustrates 7 plus frameshifts identified in the polA strain and 20 plus frameshifts in the polA107 strain. Among 7 plus frameshifts in the polA strain, one event, addition of A between nucleotides 3380–3381, was independently recovered five times. Thus, 3 sites were detected in the polA strain plus frameshifts, among which 1 was associated with a run of 4 Cs to form a run of 5 Cs and the remaining 2 were not associated with runs of bases. Among 19 plus frameshift sites recovered from the polA107 strain, 6 were associated with a run of bases. Eighteen plus frameshift sites in the polA107 strain were associated with GC clusters (Table 5). Two of 3 sites of the polA strain and 10 of 19 sites of the polA107 strain plus frameshifts were associated with imperfect inverted repeats.

View this table: In this window In a new window   Table 4. Location and types of minus frameshift mutations in a polA or polA107 strain

View this table: In this window In a new window   Table 5. Location and types of plus frameshift mutations in a polA or polA107 strain

Duplication:
Whereas duplications were not recovered in wild-type or polA1 strains, 1 duplication was counted in the polA strain and 10 duplications in the polA107 strain (Table 2). The location and types of duplication events are presented in Table 6. Among 10 duplications in the polA107 strain ranging in size from 2 to 37 bp with the majority being 2 bp in length, 3 duplications (3097–3104, 3405–3443, and 3533–3538) occurred at sites flanking repeated sequences. All the duplications, including the above 3 duplications except 3167–3168 and 3634–3635, have mutation sites with a cluster of direct repeats or imperfect inverted repeats. These sequence characteristics of duplication are essentially the same as the characteristics of frameshift and deletion mentioned above. Thus, the duplication could have been generated by slip-back misalignment of the newly synthesized fragments of nucleotides at the potentially stacked inverted repeats as suggested by IKEHATA et al. 1989 . It is thus evident that the polA107 strain is a duplication mutator.

View this table: In this window In a new window   Table 6. Location and types of duplication mutations in a polA or polA107 strain

Base substitution:
We observed nine and five base substitutions among the 51 mutations detected in KK102 and KK103, respectively, giving a base substitution rate of 15.19 x 10^-8 x 0.18 = 2.73 x 10^-8 and 9.70 x 10^-8 x 0.10 = 0.970 x 10^-8, respectively, which was essentially the same as that in the polA⁺ cells (1.91 x 10^-8 x 0.27 = 0.516 x 10^-8; Table 2). Therefore, polA and polA107 mutations do not seem to affect base substitution mutagenesis in the endogenous tonB gene. This conclusion is consistent with that of analysis of base substitution in the polA1 strain (Table 2 and AGEMIZU et al. 1999 ). Table 7 shows the location and types of base substitution mutations.

View this table: In this window In a new window   Table 7. Location and types of base substitution mutations in a polA or polA107 strain

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Using polA and polA107 alleles, together with previous results obtained in the polA1 mutant strain, we examined spontaneously occurring sequence changes in the endogenous tonB gene. It was shown that spontaneous mutations different from the wild-type strain are comprised of four different categories: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, and (4) duplications. The orders of the occurrences of these categories were different from different alleles of the polA gene. The order of occurrence by category in the polA strain was deletions, minus frameshifts, plus frameshifts, and duplications, while that in polA107 was plus frameshifts and duplications with no increases in deletions or minus frameshifts as compared to the wild-type strain. In the polA1 strain, deletions and minus frameshifts occurred predominantly in this order with no increases in duplications or plus frameshifts. Thus, there was a quite clear parallel between the categories of mutations and defects of domains in PolI, Klenow, and 5' 3' exonuclease. As summarized in Table 8, the Klenow domain is involved in preventing deletions and minus frameshifts and the 5' 3' exonuclease domain is involved in preventing duplications and plus frameshifts.

The sequence analysis in this study using the polA and polA107 strains together with our previous study using the polA1 strain (AGEMIZU et al. 1999 ) indicates a sequence specificity surrounding the mutation sites. Ten of 12 sites of deletions, 8 of 9 sites of minus frameshifts and 2 of 3 sites of plus frameshifts recovered from the polA strain were strongly associated with GC clusters. Seventeen of 19 sites of plus frameshifts and 5 of 10 sites of duplications recovered from the polA107 strain were again strongly associated with GC clusters. Furthermore, in the polA1 strain, GC clusters were seen in 18 of 20 sites of deletions and 5 of 6 sites of minus frameshifts. The GC cluster results in formation of a cluster of direct repeats as well as inverted repeats (Table 3 Table 4 Table 5 Table 6 Table 7). Thus, complex secondary structure adjoined its endpoints, and misalignment facilitated by this secondary structure may be responsible for the occurrence of deletions, frameshifts, and duplications observed in polA strains (BZYMEK and LOVETT 2001 ).

The observation that far more than half of the deletion, minus frameshift, plus frameshift, and duplication events recovered from the polA mutant strains have endpoints in the GC cluster sequence suggested that GC clusters in E. coli are quite unstable repetitive DNA sequences and may behave similarly to microsatellites found in eukaryotes and prokaryotes. As mentioned in the Introduction, microsatellite length variation is known to be associated with a number of genetic diseases including colon cancer (AALTONEN et al. 1993 ; IONOV et al. 1993 ; THIBODEAU et al. 1993 ; RICHARDS and SUTHERLAND 1994 ; ASHLEY and WARREN 1995 ). Previous studies in E. coli and yeast have shown that defects in DNA mismatch repair result in an increase in the instability of repetitive sequences (LEVINSON and GUTMAN 1987 ; STRAND et al. 1993 ). However, the observation that microsatellite instability occurs often in cells with a functional mismatch repair system suggested that other genes may contribute to prevent repetitive sequence instability. Here, we suggested that the polA⁺ gene also stabilizes repetitive sequences in E. coli. The argument further suggests two points: (1) the same mechanisms may be operating to facilitate addition or deletion of bases at tandemly repeated sequence motifs (microsatellites) and sequences without any motifs, the only difference being the frequency of occurrence, and (2) 5' 3' exonuclease of PolI processes the mismatch intermediate that will facilitate length expansion, plus frameshift and duplication, and Klenow of PolI processes the mismatch intermediate that will facilitate length shortening, minus frameshift and deletion.

PolI, encoded by the polA gene, functions in both repair and replication (see KORNBERG and BAKER 1992 ). In the absence of exogenous DNA-damaging agents, the processing of Okazaki fragments during lagging-strand synthesis is probably its main function. We have shown in this study that deletion of 5' 3' exonuclease increases the rate of sequence expansion and that of Klenow sequence shortening. Structural intermediates of repetitive sequence variation in this case may be formed during lagging-strand synthesis. It was demonstrated previously that replication-dependent frameshift mutagenesis occurred in the lagging strand (IWAKI et al. 1996 ). Thus, we propose a model that interprets processing sequence variation by PolI (Fig 2). During lagging-strand synthesis, sequence expansion occurs in the nascent DNA, which forms a mismatch bulge in the nascent DNA. When PolI binds and recognizes the mismatch in the nascent DNA, the 5' 3' exonuclease effects nascent DNA mismatch removal by flap nuclease (Fig 2A). On the other hand, sequence shortening in the nascent DNA would form a mismatch bulge in the template DNA. In this case, 3' 5' exonuclease of Klenow digests nascent DNA to solve sequence variation intermediates (Fig 2B). In this case, it is assumed that sequence expansion/shortening occurs during the DNA replication step, not during the postreplication step (see later DISCUSSION).

View larger version (22K): In this window In a new window Download PPT slide   Figure 2. Processing of length variation during lagging-strand synthesis by DNA polymerase I. The model assumes that mismatch bulges will lead to plus frameshift or duplication if present on the nascent strand (A) or to minus frameshift or deletion if present on the template strand (B).

RAD27 mutants of S. cerevisiae show a marked increase in repeat expansion (JOHNSON et al. 1995 ; KOKOSKA et al. 1998 ). The RAD27 gene encodes a 5' 3' exonuclease with structural and functional homology to the mammalian FEN1 and to the 5' 3' exonuclease domain of PolI (ROBINS et al. 1994 ). These nucleases, known to function in lagging-strand synthesis, remove the 5' flap generated by displacement synthesis by endonucleolytic cleavage (ROBINS et al. 1994 ). The mutation of 5' 3' exonuclease in E. coli leads to a marked increase in repeat expansion that also was found in RAD27 mutants. Finally, MOREL et al. 1998 demonstrated that deletion in 5' 3' exonuclease of PolI led to a marked increase in poly(AC) repeat expansion. These results are consistent with a role for this class of 5' 3' exonuclease in removing single-strand nascent DNA (flap) with mismatch intermediates.

Recently, KOKOSKA et al. 1998 and JIN et al. 2001 examined the effects on POL3 mutation of S. cerevisiae on the stability of microsatellites and found the increased rate of deletion formation. POL3 codes for DNA polymerase , which has 5' 3' DNA polymerase and 3' 5' exonuclease activities (MORRISON et al. 1991 ; SIMON et al. 1991 ). Taking together the results of RAD27 mutants and POL3 mutants, these authors proposed a model as follows: during the postreplication Okazaki fragment processing step, because of the delay to remove the terminal ribonucleotide due to RAD27 mutation, displacement of the end of the Okazaki fragment by continued synthesis from an adjacent primer results in the formation of a DNA flap that activates the 3' 5' exonuclease of DNA polymerase, producing a single-strand gap adjacent to the flap. Reassociation of the flap DNA with this region can produce a DNA molecule with unpaired repeats. The unpaired repeats on the nascent strand can lead to additions. Alternatively, the terminal ribonucleotide on the Okazaki fragment can lead to DNA synthesis block and activation of 3' 5' exonuclease without DNA flap. If a secondary structure is formed on the resulting single-strand region, subsequent synthesis across the gap can lead to a mismatch repeat on the template strand. This mismatch can lead to deletion. In this model, RAD27 and POL have antimutator roles to prevent unpaired mismatch formation during postreplication Okazaki fragment processing. TISHKOFF et al. 1997 proposed an essentially similar model. The present model presented in Fig 2 shows that 5' 3' exonuclease and Klenow activities are considered to involve the removal of polymerization errors.

The deletions, frameshifts, and duplications recovered in this study were observed within GC-rich sequences (Table 3 Table 4 Table 5 Table 6 Table 7). These repetitive sequences support the formation of secondary structures. Thus, the replication of repeats that have such a secondary structure by DNA polymerase III holoenzyme (PolIII) may lead to addition or deletion of bases. This argument is supported by the finding that frameshift mutations were found in products of in vitro DNA synthesis with weakly processive DNA polymerases or replicative DNA polymerases that lack proofreading capacities (KUNKEL 1986 ; DE BOER and RIPLEY 1988 ; BEBENEK et al. 1990 ; MO and SCHAAPER 1996 ). Thus, repetitive sequence instability may be caused by PolIII replication errors. As mentioned above, SOS induction is a microsatellite-destabilizing factor. In E. coli, three DNA polymerases, PolII (polB; BONNER et al. 1988 ), PolIV (dinB; WAGNER et al. 1999 ), and PolV (umuCD; REUVEN et al. 1999 ; TANG et al. 1999 ), are induced as part of the SOS response. NAPOLITANO et al. 2000 demonstrated that PolII and PolV are necessary and sufficient for -1 and -2 frameshift mutagenesis induced by the chemical mutagens N-2-acetylaminofluoren, respectively, and benzo()pyrene-induced -1 frameshift mutagenesis required PolIV and PolV. In that study, the dinB deletion (dinB-yafN) allele that inactivates downstream yafO and yafP (COURCELLE et al. 2001 ) was used. Thus, the evidence for PolIV is ambiguous (MCKENZIE et al. 2001 ). And thus, whether PolIII alone is involved in repetitive sequence instability or PolII, PolIV, or PolV are also involved remains to be determined.

	ACKNOWLEDGMENTS

We thank Drs. A. Nishimura (National Institute of Genetics) and C. M. Joyce (Yale University) for E. coli strains. We also thank two anonymous reviewers for comments and suggestions on the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Manuscript received June 11, 2001; Accepted for publication October 15, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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