Nucleotide excision repair in mammalia: mechanism of a primary damage recognition

Nucleotide excision repair is one of the most important pathways of DNA repair in eukaryotic cells. Defects of this system lead to serious diseases including certain kinds of cancer. Nucleotide excision repair is able to remove a wide range of the structurally diverse DNA damages such as UV induced pyrimidine dimers, bulky chemical adducts arising under the action of carcinogenic compounds or chemotherapeutical drugs on cellular DNA. A broad substrate specificity of this repair pathway is related to the main intriguing question that is the mechanism of damage recognition by the protein complex in the context of the large excess of undamaged DNA. This review is detailed on the key stage of nucleotide excision repair--the recognition of a lesion in DNA, which is still most debated. We have considered the main models of a primary damage recognition and preincision complex formation that have been suggested by the leading groups in this field. Data presented allow to suggest the model of sequential loading of the proteins of reparative complex on damage DNA as the most reasonable.     

The presence of two UvrB subunits in the UvrAB complex ensures damage detection in both DNA strands

It is generally accepted that the damage recognition complex of nucleotide excision repair in Escherichia coli consists of two UvrA and one UvrB molecule, and that in the preincision complex UvrB binds to the damage as a monomer. Using scanning force microscopy, we show here that the damage recognition complex consists of two UvrA and two UvrB subunits, with the DNA wrapped around one of the UvrB monomers. Upon binding the damage and release of the UvrA subunits, UvrB remains a dimer in the preincision complex. After association with the UvrC protein, one of the UvrB monomers is released. We propose a model in which the presence of two UvrB subunits ensures damage recognition in both DNA strands. Upon binding of the UvrA(2)B(2) complex to a putative damaged site, the DNA wraps around one of the UvrB monomers, which will subsequently probe one of the DNA strands for the presence of a lesion. When no damage is found, the DNA will wrap around the second UvrB subunit, which will check the other strand for aberrations.     

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.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

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.     

The isomerization of the UvrB-DNA preincision complex couples the UvrB and UvrC activities

In Escherichia coli nucleotide excision repair, the UvrB-DNA preincision complex plays a key role, linking adduct recognition to incision. We previously showed that the efficiency of the incision is inversely related to the stability of the preincision complex. We postulated that an isomerization reaction converts [UvrB-DNA], stable but incompetent for incision, into the [UvrB-DNA]' complex, unstable and competent for incision. Here, we identify two parameters, negative supercoiling and presence of a nick at the fifth phosphodiester bond 3' to the lesion, that accelerate the isomerization leading to an increasing incision efficiency. We also show that the [UvrB-DNA] complex is more resistant to a salt concentration increase than the [UvrB-DNA]' complex. Finally, we report that the [UvrB-DNA]' is recognized by UvrC. These data suggest that the isomerization reaction leads to an exposure of single-stranded DNA around the lesion. This newly exposed single-stranded DNA serves as a binding site and substrate for the UvrC endonuclease. We propose that the isomerization reaction is responsible for coupling UvrB and UvrC activities and that this reaction corresponds to the binding of ATP.     

抗菌肽P7抑制大肠杆菌的非膜作用机制北大核心CSCD

【目的】研究抗菌肽P7抑制大肠杆菌的非膜作用机制。【方法】P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱,分析P7与DNA的结合方式;流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响;采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列;通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响;核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。【结果】P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物,使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目,抑制大肠杆菌DNA复制。P7特异性结合rnh A使该基因表达水平显著下调2.24倍。同时,在肽的影响下参与大肠杆菌DNA复制相关的ssb、dna G、lig B和rnh A基因的表达水平显著下调(P<0.05),DNA损伤修复的rec A和rec N基因显著上调(P<0.05)。P7可降低大肠杆菌DNA和RNA的合成。【结论】P7特异性地结合rnh A序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下,参与大肠杆菌DNA复制相关的基因的表达水平下调,DNA损伤修复基因显著上调,同时抑制大肠杆菌DNA和RNA的合成。     

DNA repair triggered by sensors of helical dynamics

Nucleotide excision repair is a constitutive stress response that eliminates DNA lesions induced by multiple genotoxic agents. Unlike the immune system, which generates billions of immunoglobulins and T cell receptors for antigen recognition, the nucleotide excision repair complex uses only a few generic factors to detect an astounding diversity of DNA modifications. New data favor an unexpected strategy whereby damage recognition is initiated by the detection of abnormal oscillations in the undamaged strand opposite to DNA lesions. Another core subunit recognizes the increased susceptibility of DNA to be kinked at injured sites. We suggest that early nucleotide excision repair factors gain substrate versatility by avoiding direct contacts with modified residues and exploiting instead the altered dynamics of damaged DNA duplexes.     

UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair

The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.     

Structural basis for DNA recognition and processing by UvrB

DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.     

3-Methyladenine-DNA glycosylase (MPG protein) interacts with human RAD23 proteins

Human 3-methyladenine-DNA glycosylase (MPG protein) initiates base excision repair by severing the glycosylic bond of numerous damaged bases. In comparison, homologues of the Rad23 proteins (hHR23) and the hXPC protein are involved in the recognition of damaged bases in global genome repair, a subset of nucleotide excision repair. In this report, we show that the hHR23A and -B also interact with the MPG protein and can serve as accessory proteins for DNA damage recognition in base excision repair. Furthermore, the MPG.hHR23 protein complex elevates the rate of MPG protein-catalyzed excision from hypoxanthine-containing substrates. This increased excision rate is correlated with a greater binding affinity of the MPG protein-hHR23 protein complex for damaged DNA. These data suggest that the hHR23 proteins function as universal DNA damage recognition accessory proteins in both of these major excision repair pathways.     

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived ‘MgPar’ donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10^?4 events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption‐deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C–MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5′-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2′-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K_m and k_cat/K_m values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.