Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.     

Anti-mutagenic activity of DNA damage-binding proteins mediated by direct inhibition of translesion replication

DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication. This process is mutagenic due to the miscoding nature of the DNA lesions. We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E. coli. In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E. coli. These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication. This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.     

S-phase DNA damage checkpoint in budding yeast

Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.     

Screening for deficits in DNA repair by the response of irradiated human lymphocytes to phytohemagglutinin

An assay has been developed to measure the ability of human lymphocytes to repair damage to DNA. In this assay, purified human lymphocytes are exposed to graded doses of radiation and then stimulated with phytohemagglutinin to undergo DNA replication. The rate of incorporation of thymidine in irradiated lymphocytes during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA. In lymphocytes from normal individuals, X-irradiation with doses of 100 to 800 rads was found to inhibit phytohemagglutinin-stimulated thymidine incorporation proportionally to the dose of radiation without curtailing the induction of DNA polymerase. The response to phytohemagglutinin of lymphocytes from a patient with xeroderma pigmentosum after exposure to graded doses of X-irradiation was found to be similar to that of the normal controls, whereas the response after ultraviolet irradiation was markedly impaired. In contrast, lymphocytes from patients with ataxia telangiectasia were hypersensitive to X-irradiation. The data on these clinical syndromes support the idea that this assay measures DNA repair and indicates the feasibility of using this method for screening individuals for genetic deficits in DNA repair.     

Self and object in countertransference

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.     

Defective bypass replication of a leading strand cyclobutane thymine dimer in xeroderma pigmentosum variant cell extracts

Xeroderma pigmentosum variant (XP-V) is an inherited disorder resulting in hypersensitivity to the cytotoxic, mutagenic, and carcinogenic effects of UV light. There is evidence suggesting that XP-V cells carry a defect in the replication of UV-induced DNA damage, leading to mutations in genes, e.g., proto-oncogenes and tumor suppressor genes, of exposed skin cells. Using an in vitro assay to quantitatively evaluate replication of the most prevalent UV-derived DNA lesion, the cis,syn-thymine dimer (T x T), we have recently found that a T x T located on the leading strand can be bypassed by a bona fide human replication fork but can also induce fork uncoupling with selective synthesis of the undamaged lagging strand (D. Svoboda and J-M. Vos, Proc. Natl. Acad. Sci. USA, 92: 11975-11979, 1995). We now report the application and further refinement of this sensitive assay to the replication of a T x T-containing template by XP-V cell-free extracts. In comparison to normal controls, a 10-26-fold deficiency in the bypass replication of T x T was observed in XP-V cell extracts. In contrast, the disease extracts were as competent as controls for replication of the undamaged TT plasmid and for leading T x T-induced fork uncoupling. Besides mismatch repair and nucleotide excision repair, the bypass replication defect of XP-V may represent a novel category of hereditary mutator phenotypes affecting DNA damage processing.     

DNA damage and cell cycle checkpoints

DNA is prone to numerous forms of damage that can injure cells and impair fitness. Cells have evolved an array of mechanisms to repair these injuries. Proliferating cells are especially vulnerable to DNA damage due to the added demands of cellular growth and division. Cell cycle checkpoints represent integral components of DNA repair that coordinate cooperation between the machinery of the cell cycle and several biochemical pathways that respond to damage and restore DNA structure. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication, when the genome is replicated, and of mitosis, when the genome is segregated. Loss or attenuation of checkpoint function may increase spontaneous and induced gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair. Defects in checkpoint control have been seen in certain hereditary cancer syndromes and at early stages of cell transformation. Mutations in checkpoint control genes therefore may contribute to the genetic instability that appears to drive neoplastic evolution.     

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.     

Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function

After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication.     

DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro

Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis). Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml-2 microgram/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil. Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1-10 ng/ml). Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results help to determine the mechanism(s) through which folic acid maintains DNA stability.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

Effect of adding the topoisomerase I poison 7-ethyl-10-hydroxycamptothecin (SN-38) to 5-fluorouracil and folinic acid in HCT-8 cells: elevated dTTP pools and enhanced cytotoxicity

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.     

The dual function of the proliferating cell nuclear antigen (PCNA) in the response of human cells to UV damages

An auxiliary protein of DNA polymerases delta and epsilon, the proliferating cell nuclear antigen (PCNA), is necessary for efficient DNA replication in vivo and in vitro, and also for the repair synthesis in vitro, but its role in the excision repair of genome in vivo is not exactly established. In S-phase of unirradiated cells, PCNA is tightly bound to focal centers of DNA replication and is not removed by treatment with detergent Triton X-100, but is completely extracted from non-S-phase cells by the indicated detergent. It was shown earlier that after UV-irradiation PCNA could not be removed by the detergent even from non-S-phase cells. It was interpreted as the evidence of PCNA integration into the repair complex and of the participation of this protein in repair synthesis in vivo. In the present work the data were obtained indicating that the role of PCNA in cell response to UV-damage was not confined only to its possible involvement in repair synthesis. With the help of confocal microscopy it was established that in Triton X-100-extracted normal cells PCNA did not colocalize with the well known excision repair protein XPB/ERCC3, defective in cells from Xeroderma pigmentosum (complementation group B) patients. XPB-protein is induced by UV-irradiation in normal cells, and this induction is not observed in repair deficient cells. However, in such cells UV-light induces a detergent-resistant form of PCNA, and this form is obviously not connected with repair. It cannot be excluded that a rapid PCNA immobilization immediately after UV-irradiation of cells is needed for the facilitation of photochemical damage bypass during the subsequent replication of genome.     

Mismatch repair: mechanisms and relationship to cancer susceptibility

DNA mismatch-repair systems exist that repair mispaired bases formed during DNA replication, genetic recombination and as a result of damage to DNA. Some components of these systems are conserved in prokaryotes and eukaryotes. Genetic defects in mismatch-repair genes play an important role in common cancer-susceptibility syndromes and sporadic cancers.     

Determination of cell fate by c-Abl activation in the response to DNA damage

The cellular response to DNA damage includes growth arrest and activation of DNA repair. Certain insights into how DNA damage is converted into intracellular signals that control the genotoxic stress response have been derived from the finding that the c-Abl protein tyrosine kinase is activated by ionizing radiation and other DNA-damaging agents. c-Abl associates with the DNA-dependent protein kinase (DNA-PK) and is activated by DNA-PK-dependent phosphorylation. The ataxia telangiectasia mutated (ATM) gene product also contributes to c-Abl activation. The demonstration that c-Abl binds to p53, induces the transactivation function of p53 and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response. Interaction between c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks. Defects in G1 arrest and repair predispose to replication of damaged templates and, in the event of irreparable DNA lesions, induction of apoptosis. The available evidence indicates that c-Abl effects a proapoptotic function by a mechanism largely independent of p53. c-Abl also functions as an upstream effector of the proapoptotic JNK/SAPK and p38 MAPK pathways. In addition, c-Abl-dependent inhibition of PI 3-kinase contributes to the induction of apoptosis. The findings thus suggest that, in response to genotoxic stress, c-Abl functions in determining cell fate, that is growth arrest and repair or induction of apoptosis. The physiologic function of c-Abl may reside in control of the cellular response to DNA strand breaks that occur during DNA replication, genetic recombination and gene rearrangements.