DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

DNA damage formation and repair are tightly linked to protein–DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication (ARS1). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct interactions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.     

Chromatin and DNA damage repair

In eukaryotic cells, inheritance of both exact DNA sequence and its arrangement into the chromatin is critical for maintaining stability of the genome. Various DNA lesions induced by endogenous and exogenous factors make this maintanance problematic. To understand completely how cells resolve this problem the knowledge on the nature of these lesions, their detection, and repair within the chromatin environment should be integrated. Understanding of these processes is complicated by multiple types of DNA lesions and repair pathways, as well as the intricate organization of the chromatin. Recent advances in all these directions help to get insight on the repair regulation of DNA within the chromatin at the molecular and cellular level.     

Nucleotide excision repair: DNA damage recognition and preincision complex assembly

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells counteracting genetic changes caused by DNA damage. NER removes a wide set of structurally diverse lesions such as pyrimidine dimers arising upon UV irradiation and bulky chemical adducts arising upon exposure to carcinogens or chemotherapeutic drugs. NER defects lead to severe diseases including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to understand how a certain set of proteins recognizes various DNA lesions in the context of a large excess of intact DNA. This review focuses on DNA damage recognition and following stages resulting in preincision complex assembly, the key and still most unclear steps of NER. The major models of primary damage recognition and preincision complex assembly are considered. The contribution of affinity labeling techniques in study of this process is discussed.     

Nucleotide excision repair in higher eukaryotes: Mechanism of primary damage recognition

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation, and bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to severe diseases, including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to study how a certain set of proteins recognizes DNA lesions in contest of a large excess of intact DNA. The review focuses on DNA damage recognition, the key and, as yet, most questionable step of NER. The main models of primary damage recognition and preincision complex assembly are considered. The model of a sequential loading of repair proteins on damaged DNA seems most reasonable in light of the available data.     

Shaping chromatin for repair

To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA–RPA and XPC–RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSβ were sensitive to psoralen ICLs. To further investigate the role of MutSβ in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSβ and NER proteins with Tdp-ICLs. We found that MutSβ bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSβ interacted with XPA–RPA or XPC–RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.     

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.     

Chromatin challenges during DNA replication and repair

Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic landscape may be stably maintained even in the face of dramatic changes in chromatin structure.     

Organization of DNA damage,excision repair,and mutagenesis in chromatin: A genomic perspective

Genomic DNA is constantly assaulted by both endogenous and exogenous damaging agents. The resulting DNA damage, if left unrepaired, can interfere with DNA replication and be converted into mutations. Genomic DNA is packaged into a highly compact yet dynamic chromatin structure, in order to fit into the limited space available in the nucleus of eukaryotic cells. This hierarchical chromatin organization serves as both the target of DNA damaging agents and the context for DNA repair enzymes. Biochemical studies have suggested that both the formation and repair of DNA damage are significantly modulated by chromatin. Our understanding of the impact of chromatin on damage and repair has been significantly enhanced by recent studies. We focus on the nucleosome, the primary building block of chromatin, and discuss how the intrinsic structural properties of nucleosomes, and their associated epigenetic modifications, affect damage formation and DNA repair, as well as subsequent mutagenesis in cancer.