Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.     

In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC+ x i ets 1 segCDelta crosses, where ets 1 is an insertion in the initial part of the rII gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets 1, and segCDelta is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets 1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets 1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i x i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets 1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.     

Marker-Dependent Recombination in T4 Bacteriophage. II. the Evaluation of Mismatch Repairabilities in Crosses within Indicator Distances

The contribution of mismatch repair to genetic recombination in T4 phage has been evaluated by three independent approaches: (1) testing for non-additivity of recombinant frequencies; (2) measurements of double exchange frequencies in three-factor crosses: (3) comparisons of recombination abilities of mutations occupying the same site. Quantitative agreement among the results of these approaches suggests that within distances much less than the mean length of hybrid regions, mismatch repair accounts perfectly for high negative interference as measured in three-factor crosses and as manifested by non-additivity in two-factor crosses. The mismatch repair mechanism readily recognizes only particular mismatches, the repair frequency being dependent on the base sequence in both strands of the mismatched region.     

Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.     

Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes.

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.     

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.     

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49. In the present paper, we tested this hypothesis in vivo. We used an experimental model system that combines site-specific double-strand breaks with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. We compared recombination of homoallelic HR and LR markers in the S17 and S17 E727 background (amber mutations in the uvsX and in the uvsX and 49 genes, respectively). In S17-crosses, the HR and LR markers retain their respective high-recombination and low-recombination behavior. In S17 E727-crosses, however, the HR and LR markers show no difference in the recombination frequency and both behave as LR markers. We conclude that endo VII is the enzyme that recognizes mismatches in recombinational heteroduplexes and performs their incision. This role for endo VII was suggested previously from biochemical studies, but this is its first in vivo demonstration.     

Spontaneous Mutation in the Escherichia Coli Laci Gene

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.     

Functional mutation of DNA polymerase beta found in human gastric cancer--inability of the base excision repair in vitro.

DNA polymerase beta (polbeta) is one of mammalian DNA polymerases and is known to be involved in a G:T/G:U mismatch repair. In order to investigate an involvement of this enzyme in a base excision repair, we searched a mutation of human polbeta in human gastric cancer and studied a function of the mutation. We observed cancer-specific missense mutations in 6 of 20 samples. All of these mutations were, however, heterozygous. We further analyzed the base excision repair activity of these mutants to know whether these mutants cause an error of mismatch repair. One of these mutants, which resulted in an amino acid substitution of Glu for Lys at codon 295, showed an inhibitory effect by in vitro base excision repair assay, suggesting that this mutation might play some role in carcinogenesis of the gastric mucosa.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene.

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.     

Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4

We have mapped the mutants isolated by Nelson et al. (1981) that reduce the amount of rIIB protein synthesized during bacteriophage T4 infection of Escherichia coli B and characterized their rIIB expression in vivo. These mutants fall into four distinct groups in terms of mapping and phenotype. We have located the probable site of each mutation on the DNA sequence. We have also analyzed a number of other mutations near the initiating AUG of rIIB with respect to their rIIB expression. In some of these mutants, ribosomal recognition of the wild-type initiating AUG is precluded and so initiation occurs at a different AUG, which, in some instances, we have identified.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.     

Recombination in phage T4 gene-43 (DNA polymerase) mutants.

Summary The effect of phage T4 gene 43 (DNA polymerase) mutations on recombination between adjacent base pairs was measured in rII amber and opal mutants.The mutator allele tsL56 did not promote recombination frequencies at the two sites in which its effect was studied. The antimutator allele tsCB87 caused slight or no reduction in recombination frequencies at five sites.Abbreviations A, T, G and C are adenine, thymine, guanine and 5-hydroxymethylcytosine, respectively     

Characterization of components of the mismatch repair machinery in Trypanosoma brucei

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base-base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.     

Targeted gene repair and its application to neurodegenerative disorders

Synthetic DNA oligonucleotides can direct the exchange of single nucleotides within coding regions of mammalian genes by hybridizing to their complementary sequence in the chromosome and creating a recombination joint structure with a single mismatched base pair. Inherent DNA repair processes recognize the mismatch and resolve it using the DNA sequence of the oligonucleotide vector as the template. This gene surgery approach can be used to repair mutations or to disrupt tri-nucleotide repeats in dysfunctional genes responsible for neurological disorders.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.