Single-stranded DNA gaps, tails and loops are repaired in Escherichia coli

Uniformly methylated heteroduplex plasmids which contained 6 mismatched regions, including loops of 24, 30, 248 and 283 nucleotides, as well as single-stranded gaps and free ends were introduced into a recombination-deficient strain of bacteria, and the products of repair were analyzed. The results indicate that these cells are capable of repairing all of these structures, although with different efficiencies. Repair of single-stranded gaps and free ends, which occurs most efficiently, is always associated with acquisition of information from the uncut strand (unidirectional repair). Regions containing single loops or twin loops were repaired at similar efficiencies. In these cases each of the two strands was capable of acting as the template for repair (bidirectional repair). At sites containing twin or substitution loops, the larger of the loops was removed twice as efficiently as the smaller loop. DNA sequencing of the repaired regions indicated that the repair is precise. The data also suggest that markers separated by only 58 nucleotides do not always segregate together indicating that repair tracts may be relatively short.     

Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha,a chemical inhibitor of p53

We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.     

Ku binds telomeric DNA in vitro.

Ku is a heterodimeric protein with high binding affinity for ends, nicks, and gaps in double-stranded DNA. Both in mammalian cells and in budding yeast, Ku plays a role in nonhomologous end joining in the double strand break repair pathway. However, Ku has a more significant role in DNA repair in mammalian cells compared with yeast, in which a homology-dependent pathway is the predominant one. Recently Ku has been shown to be a likely component of the telomeric complex in yeast, suggesting the possibility of a similar role for Ku at mammalian telomeres. However, long single-stranded G-rich overhangs are continuously present at mammalian but not at yeast telomeres. These overhangs have the potential to fold in vitro into G-G base-paired conformations, such as G-quartets, that might prevent Ku from recognizing telomeric ends and thus offer a mechanism to sequester the telomere from the prevalent double strand break repair pathway in mammals. We show here that Ku binds to mammalian telomeric DNA ends in vitro and that G-quartet conformations are unable to prevent Ku from binding with high affinity to the DNA. Our results indicate that the DNA binding characteristics of Ku are consistent with its direct interaction with telomeric DNA in mammalian cells and its proposed role as a telomere end factor.     

Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop

Using heteroduplex molecules formed from a pair of plasmids, one of which contains a small deletion relative to the other, it is shown that bacterial topoisomerase I can relax a positively supercoiled DNA if a short single-stranded loop is placed in the DNA. This result supports the postulate that the specificity of bacterial DNA topoisomerase I for negatively supercoiled DNA in its relaxation reaction derives from the requirement of a short single-stranded DNA segment in the active enzyme-substrate complex. Nucleolytic and chemical probing of complexes between bacterial DNA topoisomerase I and heteroduplex DNA molecules containing single-stranded loops ranging from 13 to 27 nucleotides in length suggests that the enzyme binds specifically to the region containing a single-stranded loop; the site of DNA cleavage by the topoisomerase appears to lie within the single-stranded loop, with the enzyme interacting with nucleotides on both sides of the point of cleavage.     

Interaction of human replication protein A with DNA-duplexes, containing gaps of varying sizes

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3' end of a gap-flanking oligonucleotide with DNA polymerase beta, and an oligonucleotide containing a 5'-photoreactive group was chemically synthesized. The 5' end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3' end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA-DNA interaction, which has been advanced earlier.     

Size and frequency of gaps in newly synthesized DNA of xeroderma pigmentosum human cells irradiated with ultraviolet light

Native newly synthesized DNA from human cells (xeroderma pigmentosum type) irradiated with ultraviolet light releases short pieces of DNA (L-DNA) when incubated with the single-strand specific S1 nuclease. This is not observed in the case of unirradiated cells. Previous experiments had shown that the L-DNA resulted from the action of S1 nuclease upon gaps, i.e., single-stranded DNA discontinuities in larger pieces of double-stranded DNA. We verified that the duplex L-DNA, that arises from the inter-gap regions upon S1 nuclease treatment, has a size which approximates the distance between two pyrimidine dimers on the same strand; this has been observed at different fluences of ultraviolet-light and indicates that the gap is related to or opposite the dimer. A method was devised to measure the size of the gaps. A Poisson distribution analysis of the percentage of the L-DNA produced as a function of S1 nuclease concentration made this possible. 65% of the gaps corresponded to stretches of 1,250 nucleotides and 35% to stretches of 150 nucleotides. These parameters have been considered in the proposition of a model for DNA synthesis on a template containing pyrimidine dimers.     

Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases.

The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA. We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 3'-->5' exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3' terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.     

Repair of gaps opposite lesions by homologous recombination in mammalian cells

Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability.     

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.     

Repair of DNA damage in shuttle vectors, virus, and chromosomal DNAs may depend on their biological imprinting--a 'Pygmalion' effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli.

The fate of heteroduplex molecules containing 5-, 7-, 9-, 192-, 410-, and 514-base loops after transformation of wild-type and various mutant strains of Escherichia coli has been examined. No evidence for repair was obtained for the wild type or for strains with mutations in the following genes: mutS, recA, recBC sbcBC, recD, recF, recJ, recN, recO, recR, recBC sbcBC recF uvrA, recG ruvC, ruvB, lexA3, lexA51, uvrA, nfo xth nth, polA(Ts), or pcnB. These results rule out the involvement of the SOS system and most known recombination and repair pathways. Repair of heteroduplex molecules containing 410- and 514-base loops was observed when a 1-base deletion-insertion mismatch was present nearby. The repair of both the mismatch and the loops was directed by the state of dam methylation of the DNA chains and was dependent on the product of the mutS gene. A high efficiency of repair (95%) was found even when the mismatch and the loops were 1,448 nucleotides apart. We conclude that multibase loops in DNA can be removed only as a consequence of corepair by dam-directed mismatch repair.     

The Large Subunit of Replication Factor C (Rfc1p/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces Cerevisiae

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.     

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.     

Replication of DNA in mammalian chromosomes

A study of sedimentation and buoyant density of Okazaki fragments from mammalian chromosomes along with electron microscopic studies indicate that fragments from about 200 to 1200 nucleotides long may have RNA segments covalently attached. The fragments in some CsCl isopycnic gradients banded in two rather distinct bands. One band corresponds to the density of single-stranded DNA, but the other has a higher buoyant density which could be conferred by a segment of RNA up to 180 nucleotides or more in length. The RNA was not removed by denaturing conditions which separated DNA strands consisting of several thousand nucleotide pairs. When the material of higher buoyant density was spread for electron microscopy under conditions which would extend single-stranded DNA chains, but leave RNA in a coil or bush the chains with a higher buoyant density usually had a bush attached at one end. Under conditions that were thought to favor gap filling over chain elongation near growing forks, the DNA produced by pulse labeling with bromodeoxyuridine had a buoyant density which would indicate substitution to about 15 percent in one chain. If this substitution represents filling of gaps occupied by RNA before the pulse, the segments would be about 180 nucleotides in length assuming about 1,000 nucleotides between each segment.     

Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to [3H]thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.     

Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision

Synthetic DNA linkers containing a single mismatched nucleotide (C:A) are repaired without bias at high efficiency when introduced into mammalian cells on a SV40 shuttle vector. From the pattern of repair in vectors containing multiple linkers, it appears that DNA synthesis following mismatch excision can replace a length of DNA as short as 40 nucleotides. Furthermore, results from the introduction of linker molecules containing combinations of single-strand nicks suggest that transient unsealed nicks do not drive the direction of mismatch repair in mammalian cells, as has previously been proposed.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.     

Novel PMS1 alleles preferentially affect the repair of primer strand loops during DNA replication

Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.