The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.

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Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

Replication protein A [RPA; also known as replication factor A (RFA) and human single-stranded DNA-binding protein] is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination. RPA homologues have been identified in all eukaryotic organisms examined and are all abundant heterotrimeric proteins composed of subunits of approximately 70, 30, and 14 kDa. Members of this family bind nonspecifically to single-stranded DNA and interact with and/or modify the activities of multiple proteins. In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage). Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell. RPA may also have a role in modulating gene expression.

REPLICATION PROTEIN A: A Heterotrimeric, Single-Stranded DNA-Binding Protein Required for Eukaryotic DNA Metabolism

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.

DNA mismatch repair

DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSalpha and MutLalpha, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.

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Mechanisms and functions of DNA mismatch repair

From its very beginning, life has faced the fundamental problem that the form in which genetic information is stored is not chemically inert. DNA integrity is challenged by the damaging effect of numerous chemical and physical agents, compromizing its function. To protect this Achilles heel, an intricate network of DNA repair systems has evolved early in evolution. One of these is nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway that counteracts the deleterious effects of a multitude of DNA lesions, including major types of damage induced by environmental sources. The most relevant lesions subject to NER are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), two major kinds of injury produced by the shortwave UV component of sunlight. In addition, numerous bulky chemical adducts are eliminated by this process. Within the divergent spectrum of NER lesions, significant distortion of the DNA helix appears to be a common denominator. Defects in NER underlie the extreme photosensitivity and predisposition to skin cancer observed with the prototype repair syndrome xeroderma pigmentosum (XP). Seven XP complementation groups have been identified, representing distinct repair genes XPA–G (discussed in detail below). In the last decade, all key NER factors have been cloned and the core of the ‘cut-and-paste’ reaction has been reconstituted in vitro from purified components. Recently, XPC (complexed to hHR23B) has been identified as a DNA-damage sensor and repair-recruitment factor. The general transcription factor complex TFIIH, containing the XPB and XPD helicases, mediates strand separation at the site of the lesion. XPA verifies the damage in an open DNA conformation and is crucial in the assembly of the remainder of the repair machinery. Replication protein A (RPA) stabilizes the opened DNA complex and is involved in positioning the XPG and ERCC1–XPF endonucleases responsible for the DNA incisions around the lesion. After removal of the damagecontaining oligonucleotide, typically 24–32 nucleotides in length, general replication factors fill in the remaining gap and close it. Two modes of NER can be distinguished: repair of lesions over the entire genome, referred to as global genome NER (GG–NER), and repair of transcription-blocking lesions present in transcribed DNA strands, hence called transcription-coupled NER (TC–NER). Most XP groups harbor defects in a common component of both NER subpathways. GG–NER is dependent on the activity of all factors mentioned above, including the GG– NER-specific complex XPC–hHR23B. The rate of repair for GG–NER strongly depends on the type of lesion. For instance, 6-4PPs are removed much faster from the genome than CPDs, probably because of differences in affinity of the damage sensor XPC–hHR23B. In addition, the location (accessibility) of a lesion influences the removal rate in vivo. In TC–NER, damage is detected by the elongating RNA polymerase II complex when it encounters a lesion. Interestingly, a distinct disorder, Cockayne syndrome (CS), is associated with a specific defect in transcription-coupled repair. The identification of two complementation groups (CS-A and CS-B) shows that at least two gene products are specifically needed for fast and efficient repair of transcribed strands. Phenotypically, CS is a very pleiotropic condition characterized by photosensitivity as well as severe neurological, developmental, and premature aging features. Most of these symptoms are not seen even with totally NERdeficient XP patients. The additional symptoms of CS suggest that transcription-coupled repair and/or the CS proteins have functions beyond NER. Also, non-NERspecific lesions (such as oxidative damage) that stall transcription elongation appear to be removed in a transcription-coupled fashion, linking a blocked polymerase to multiple repair pathways. Intriguingly, some XP-B, XP-D, and XP-G patients display CS features combined with XP manifestations. Yet other XP-B and XP-D individuals suffer from the CS-like brittle-hair syndrome trichothiodystrophy (TTD). This clinical conundrum points to additional roles of these NER factors as well. A recent mouse model for TTD has linked mutations in the XPD subunit of the dual functional TFIIH complex with deficiencies in basal transcription underlying at least some of the TTD manifestations. Thus, NER defects are associated with a surprisingly wide clinical heterogeneity due to additional functions of the NER factors involved. 1Corresponding author. E-MAIL Hoeijmakers@gen.fgg.eur.nl; FAX 31 10 408 9468.

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Molecular mechanism of nucleotide excision repair

Reconstitution of yeast nucleotide excision repair with purified rad proteins, replication protein a, and transcription factor tfiih

Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair

Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3

IN eukaryotes nucleotide excision repair of DNA damaged by ultraviolet radiation requires several gene products; defects in this process result in the cancer-prone syndrome xeroderma pigmento-sum (XP) in humans1,2. TheRAD2 gene is one of at least seven genes indispensable for excision repair in the yeast Saccharomyces cerevisiae2, and its encoded protein shares remarkable homology with the XP group-G gene product3. Here we overproduce the RAD2-encoded protein in S. cerevisiae, purify it to near homogeneity, and show that RAD2 protein in the presence of magnesium degrades circular single-stranded DNA. The RAD2 endonuclease is specific for single-stranded DNA as it does not act on doublestranded DNA. Given the absolute requirement for RAD2 in the incision step of excision repair, our findings directly implicate RAD2 protein and its human homologue XPG protein as a catalytic component that incises the damaged DNA strand during excision repair. Furthermore, our results indicate that eukaryotes probably employ two distinct endonuclease activities to mediate the dual incision at the damage site.

Yvette Habraken

Papers

Reconstitution of yeast nucleotide excision repair with purified rad proteins, replication protein a, and transcription factor tfiih

Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair

Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3

Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease

ATP-dependent Assembly of a Ternary Complex Consisting of a DNA Mismatch and the Yeast MSH2-MSH6 and MLH1-PMS1 Protein Complexes