The role of deoxyribonucleotide metabolism in 5-bromo-2′-deoxyuridine mutagenesis in mammalian cells

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and siste-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrURA residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.     

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

Summary To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34° C). One such marker, rII SN103, which showed the highest stimulation at 34° C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34° C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34° C cultures showed no significant deviations from values obtained at 30° C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the imitator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.     

Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli

Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.     

Imbalanced deoxyribonucleoside triphosphate pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections

DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. Since other factors may be involved, it is important to identify specific mutations induced by specific pool imbalances. Using bacteriophage T4, we have developed a system for such analysis. We prepare double mutants of T4; one mutation affects a phage-coded enzyme of deoxyribonucleoside triphosphate (dNTP) metabolism, while the second is an rII mutation known to revert along a specific pathway. We determine dNTP pools in infection by such a mutant and measure both the spontaneous reversion rate of the rII mutation and, in some cases, the nucleotide sequence at the mutant site. In this paper we analyze mutations induced by a deficiency of T4-encoded deoxycytidylate deaminase. This causes pools of 5-hydroxymethyl-dCTP to expand some 30-fold, while dTTP pools contract. This specifically stimulates AT-to-GC reversion. One of the four AT-to-GC reverters tested, rIIUV215, increases its reversion rate at least 1000-fold under these pool-imbalance conditions, while the other mutants tested show increases of only about 10-fold. Therefore, factors other than dNTP competition, including local DNA sequence environment, must be invoked to fully explain mechanisms of dNTP pool imbalance-induced mutagenesis. We discuss models for this, and we also report unexpected effects of the dCMP deaminase deficiency upon pools of ribonucleoside triphosphates.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.     

Polynucleotide Phosphorylase Plays an Important Role in the Generation of Spontaneous Mutations in Escherichia coli

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif^r and the gyrB gene leading to Nal^r and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.     

Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint

A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is one of the key prerequisites for faithful genome duplication. Both the overall concentration and the balance among the individual dNTPs (dATP, dTTP, dGTP, and dCTP) are tightly regulated, primarily by the enzyme ribonucleotide reductase (RNR). We asked whether dNTP pool imbalances interfere with cell cycle progression and are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. By introducing single amino acid substitutions in loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR, we obtained a collection of strains with various dNTP pool imbalances. Even mild dNTP pool imbalances were mutagenic, but the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity. The S-phase checkpoint was activated by the depletion of one or several dNTPs. In contrast, when none of the dNTPs was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression.     

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.     

dNTP pools determine fork progression and origin usage under replication stress

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.     

ATP:AMP phosphotransferase from baker''s yeast. Purification and properties

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and maximum deoxyribonucleoside 5'-triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2-3 pmol/10(6) cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP leads to dNDP leads to dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 micrograms 5-fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5-Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.     

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

Implications for a reduced DNA-elongation rate in polyamine-depleted cells

Treatment of Ehrlich ascites tumor cells with 2-difluoromethylornithine (F2MeOrn), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, resulted in depleted putrescine and spermidine content, and reduced growth rate. We have previously shown that adenine ribonucleotide levels are substantially increased in these polyamine-depleted cells. The present paper addresses the question whether the elevated ATP pool is accompanied by a concomitant increase in the dATP pool. If this is the case, the observed growth inhibition could be explained by the well-known dATP-mediated feedback inhibition of ribonucleotide reductase. We found that dNTP pools were not unbalanced and that dNTP synthesis was not arrested in polyamine-depleted cells. Moreover, the dNTP content and the activity of ribonucleotide reductase (CDP reduction) and thymidylate synthase, remained elevated despite the fact that the cells were inhibited in their growth by F2MeOrn treatment. Incorporation of a radiolabeled precursor into DNA was initially lower in F2MeOrn-treated. cells than in control cells. However, while incorporation of a radiolabeled precursor into DNA decreased markedly in plateau-phase control cells, it remained at a higher level in cells inhibited in growth by polyamine depletion. This discrepancy may be explained by the fact that polyamine-depleted cells accumulated in the S phase, and that they had an increased content of acid-soluble radiolabeled DNA precursor. Our data indicate that polyamine depletion adversely affects the DNA synthetic machinery by reducing the rate of elongation.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Curing the Megaplasmid pTT27 from Thermus thermophilus HB27 and Maintaining Exogenous Plasmids in the Plasmid-Free Strain

Stepwise deletions in the only plasmid in Thermus thermophilus HB27, megaplasmid pTT27, showed that two distantly located loci were important for maintenance of the plasmid. One is a minimum replicon including one gene, repT, coding a replication initiator, and the other encodes subunits of class I ribonucleotide reductase (RNR) for deoxynucleoside triphosphate (dNTP) synthesis. Since the initiator protein, RepT, bound to direct repeats downstream from its own gene, it was speculated that a more-downstream A+T-rich region, which was critical for replication ability, could be unwound for replication initiation. On the other hand, the class I RNR is not necessarily essential for cell growth, as evidenced by the generation of the plasmid-free strain by the loss of pTT27. However, the plasmid-free strain culture has fewer viable cells than the wild-type culture, probably due to a dNTP pool imbalance in the cell. This is because of the introduction of the class I RNR genes or the supplementation of 5′-deoxyadenosylcobalamin, which stimulated class II RNR encoded in the chromosome, resolved the decrease in the number of viable cells in the plasmid-free strain. Likewise, these treatments dramatically enhanced the efficiency of transformation by exogenous plasmids and the stability of the plasmids in the strain. Therefore, the class I RNR would enable the stable maintenance of plasmids, including pTT27, as a result of genome replication normalized by reversing the dNTP pool imbalance. The generation of this plasmid-free strain with great natural competence and its analysis in regard to exogenous plasmid maintenance will expand the availability of HB27 for thermophilic cell factories.     

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ~1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.     

Collapse and repair of replication forks in Escherichia coli

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.