Non-Catalytic Domains of DNA Polymerase λ: Influence on Enzyme Activity and Its Regulation.

DNA polymerase λ (Polλ) belongs to the same structural X-family as DNA polymerase ß, the main polymerase of base excision repair. The role of Polλ in this process remains not fully understood. A significant difference between the two DNA polymerases is the presence of an extended non-catalytic N-terminal region in the Polλ structure. The influence of this region on the interaction of Polλ with DNA and multifunctional proteins, poly(ADP-ribose)polymerase 1 (PARP1) and replication protein A (RPA), was studied in detail for the first time. The data obtained suggest that non-catalytic Polλ domains play a suppressor role both in relation to the polymerase activity of the enzyme and in interaction with DNA and PARP1.

[Interactome of Base and Nucleotide Excision DNA Repair Systems].

The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in the case of BER and bulky damage caused by UV radiation or chemical carcinogens in the case of NER. In some cases, however, the repair process follows a more complex scenario, which implies that the repair pathways exchange proteins and interact with each other to form a common interactome. This review describes the BER and NER mechanisms and discusses the current data on the involvement of the NER proteins in the repair of DNA lesions caused by oxidative stress and the BER proteins in the removal of bulky DNA adducts. We also discuss the role of poly(ADP-ribose) polymerase 1 in the regulation of the BER and NER processes and their coordination in the repair of complex (cluster) lesions.

Post-translational Modifications of Nucleotide Excision Repair Proteins and Their Role in the DNA Repair.

Nucleotide excision repair (NER) is one of the major DNA repair pathways aimed at maintaining genome stability. Correction of DNA damage by the NER system is a multistage process that proceeds with the formation of multiple DNA-protein and protein-protein intermediate complexes and requires precise coordination and regulation. NER proteins undergo post-translational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and poly(ADP-ribosyl)ation. These modifications affect the interaction of NER factors with DNA and other proteins and thus regulate either their recruitment into the complexes or dissociation from these complexes at certain stages of DNA repair, as well as modulate the functional activity of NER proteins and control the process of DNA repair in general. Here, we review the data on the post-translational modifications of NER factors and their effects on DNA repair. Protein poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerase 1 and its impact on NER are discussed in detail, since such analysis has not been done before.

[Replication protein A as a major eukaryotic single-stranded DNA-binding protein and its role in DNA repair].

Replication protein A (RPA) is a key regulator of eukaryotic DNA metabolism. RPA is a highly conserved heterotrimeric protein and contains multiple oligonucleotide/oligosaccharide-binding folds. The major RPA function is binding to single-stranded DNA (ssDNA) intermediates forming in DNA replication, repair, and recombination. Although binding ssDNA with high affinity, RPA can rapidly diffuse along ssDNA and destabilizes the DNA secondary structure. A highly dynamic RPA binding to ssDNA allows other proteins to access ssDNA and to displace RPA from the RPA-ssDNA complex. As has been shown recently, RPA in complex with ssDNA is posttranslationally modified in response to DNA damage. These modifications modulate the RPA interactions with its protein partners and control the DNA damage signaling pathways. The review considers up-to-date data on the RPA function as an active coordinator of ssDNA intermediate processing within DNA metabolic pathways, DNA repair in particular.

Interaction of Nucleotide Excision Repair Protein XPC-RAD23B with DNA Containing Benzo[a]pyrene-Derived Adduct and Apurinic/Apyrimidinic Site within a Cluster.

The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms - NER and BER, respectively. Interaction of NER protein XPC-RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N(2)-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC-RAD23B crosslinks to DNA containing (+)-trans-BPDE-N(2)-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N(2)-dG isomers depends on the AP site position in the opposite strand. The influence of XPC-RAD23B on hydrolysis of an AP site clustered with BPDE-N(2)-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC-RAD23B was shown to stimulate the endonuclease and inhibit the 3'-5' exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.

[Tyrosyl-DNA Phosphodiesterase 1 Is a New Player in Repair of Apurinic/Apyrimidinic Sites].

Genomic DNA is constantly damaged by the action of exogenous factors and endogenous reactive metabolites. Apurinic/apyrimidinic sites (AP sites), which occur as a result of DNA glycosylase induced or spontaneous hydrolysis of the N-glycosidic bonds, are the most common damages of DNA. The chemical reactivity of AP sites is the cause of DNA breaks, and DNA-protein and DNA-DNA crosslinks. Repair of AP sites is one of the most important mechanisms for maintaining genome stability. Despite the fact that the main participants of the AP site repair are very well studied, the new proteins that could be involved potentially in this process as "back up" players or perform certain specialized functions are being found. This review is dedicated to one of these proteins, tyrosyl-DNA phosphodiesterase 1 (Tdp1), for which we have recently shown that in addition to its main activity of specific cleavage of the tyrosyl-DNA bond formed via a covalent attachment of topoisomerase 1 (Top1) to DNA, Tdp1 is able to initiate the cleavage of the internal AP sites in DNA and their following repair. Tdp1 was discovered in Saccharomyces cerevisiae yeast as an enzyme hydrolyzing the covalent bond between tyrosyl residue of topoisomerase 1 and 3'-phosphate group in DNA. Tdp1 is the major enzyme which carries out the repair of the irreversible complexes of DNA and topoisomerase 1, which appear. in the presence of Top 1 inhibitors, such as camptothecin, therefore Tdp1 is a very important target for the development of inhibitors--anticancer drugs. Besides, Tdp1 hydrolyzes a wide range of 3'-terminal DNA modifications and the 3'-end nucleosides and its derivatives to form a 3'-phosphate. Tdp1 ability to cleave AP sites suggests its involvement in the base excision repair as an alternative enzyme to cleave AP sites instead of AP endonuclease 1--the major enzyme hydrolyzing AP sites in DNA repair process.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E >> K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

[Repair of bulky DNA damages--derivatives of polycyclic aromatic hydrocarbons].

The genomic DNA is damaged under the influence of different environmental factors such as air pollutions, ultraviolet and ionizing radiation, and toxic substances that negatively impact on the humans. Air pollution by the products of incomplete combustion of hydrocarbon fuels and waste of various industries are main sources of polycyclic aromatic hydrocarbons. Some metabolites of these compounds can damage DNA through forming the bulky DNA adducts that potentially leads to mutagenesis and cancer. A nucleotide excision repair is the major pathway for the reparation of such DNA lesions in eukaryotic cells. The excision efficiency of bulky adducts depends on many factors including the structure of a substituent and degree of DNA double helix distortion induced by a lesion. The most danger for cell is clustered DNA lesions. To repair them the cooperation of different DNA repair systems is required in the process of damage recognition and removal. This review is focused on the features of repair mechanisms for DNA with bulky lesions appeared in the result of action of natural carcinogen benzo[a]pyrene as an example.

Influence of centrin 2 on the interaction of nucleotide excision repair factors with damaged DNA.

We have examined the influence of centrin 2 (Cen2) on the interaction of nucleotide excision repair factors (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes bearing the dUMP derivative 5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate. The fluorescein residue linked to the nucleotide base imitates a bulky lesion of DNA. Cen2 stimulated the binding and increased the yield of DNA adducts with XPC-HR23b, a protein recognizing bulky damages in DNA. Stimulation of the binding was most pronounced in the presence of Mg(2+) and demonstrated a bell-shaped dependence on Cen2 concentration. The addition of Cen2 changed the stoichiometry of RPA-DNA complexes and diminished the yield of RPA-DNA covalent crosslinks. We have shown that Cen2 influences the binding of RPA and XPA with DNA, which results in formation of additional DNA-protein complexes possibly including Cen2. We have also found some evidence of direct contacts between Cen2 and DNA. These results in concert with the literature data suggest that Cen2 can be a regulatory element in the nucleotide excision repair system.

Interaction of nucleotide excision repair proteins with DNA containing bulky lesion and apurinic/apyrimidinic site.

The interaction of nucleotide excision repair (NER) proteins (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes containing the bulky lesion-mimicking fluorescein-substituted derivative of dUMP (5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate) in a cluster with a lesion of another type (apurinic/apyrimidinic (AP) site) has been studied. It is shown that XPC-HR23b is modified to a greater extent by the DNA duplex containing an AP site opposite nucleotide adjacent to the fluorescein residue than by DNA containing an AP site shifted to the 3'- or 5'-end of the DNA strand. The efficiency of XPA modification by DNA duplexes containing both AP site and fluorescein residue is higher than that by DNA lacking the bulky lesion; the modification pattern in this case depends on the AP site position. In accordance with its major function, RPA interacts more efficiently with single-stranded DNA than with DNA duplexes, including those bearing bulky lesions. The observed interaction between the proteins involved in nucleotide excision repair and DNA structures containing a bulky lesion processed by NER and the AP site repaired via base excision repair may be significant for both these repair pathways in cells and requires the specific sequence of repair of clustered DNA lesions.

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 in global genome repair.

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells that counteract the formation of genetic damage. 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 understand how a certain set of proteins recognizes various DNA lesions in the contest of a large excess of intact DNA. This review focuses on DNA damage recognition, the key and, as yet, most questionable step of NER. Understanding of mechanism of this step of NER may give a key contribution to study of similar processes of DNA damage recognition (base excision repair, mismatch repair) and regulation of assembly of various DNA repair machines. The major models of primary damage recognition and pre-incision complex assembly are considered. The model of a sequential loading of repair proteins on damaged DNA seems most reasonable in the light of the available data. The possible contribution of affinity labeling technique in study of this process is discussed.

Interaction of DNA topoisomerase 1 with DNA intermediates and proteins of base excision repair.

The interaction of human recombinant DNA topoisomerase 1 (Top1) with linear and circular DNA structures containing a nick or short gap but lacking a specific Top1 recognition site was studied. The effect of key excision repair proteins on formation of the Top1 covalent adduct with the DNA repair intermediates was shown. Partial inhibition of the Top1-DNA-adduct formation upon addition of poly(ADP-ribose) polymerase 1 in the absence of NAD+ was shown, whereas in the presence of NAD+ formation of a high molecular weight product, most likely corresponding to poly(ADP)-ribosylated Top1-DNA adduct, was observed. The data show that the key base excision repair proteins can influence formation of suicide Top1-DNA adducts. Top1 was identified by immunoprecipitation in the bovine testis nuclear extract as the protein forming the main modification product with nick-containing DNA.

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA.

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.

[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.

DNA polymerases beta and lambda as potential participants of TLS during genomic DNA replication on the lagging strand.

The main strategy used by pro- and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases beta and lambda on DNA substrates using mono- or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg2+ or Mn2+. DNA polymerases beta and lambda were able to catalyze DNA synthesis across THF only in the presence of Mn2+. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase beta. As for DNA polymerase lambda, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase beta. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase lambda. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases beta and lambda on DNA templates containing damage.

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages.

Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5 -end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.

Analysis of interactions of DNA polymerase beta and reverse transcriptases of human immunodeficiency and mouse leukemia viruses with dNTP analogs containing a modified sugar residue.

Substrate properties of various morpholinonucleoside triphosphates in the reaction of DNA elongation catalyzed by DNA polymerase beta, reverse transcriptase of human immunodeficiency virus (HIV-1 RT), and reverse transcriptase of Moloney murine leukemia virus (M-MuLV RT) were compared. Morpholinonucleoside triphosphates were utilized by DNA polymerase beta and HIV-1 reverse transcriptase as substrates, which terminated further synthesis of DNA, but were virtually not utilized by M-MuLV reverse transcriptase. The kinetic parameters of morpholinoderivatives of cytosine (MorC) and uridine (MorU) were determined in the reaction of primer elongation catalyzed by DNA polymerase beta and HIV-1 reverse transcriptase. MorC was a more effective substrate of HIV-1 reverse transcriptase and significantly less effective substrate of DNA polymerase beta than MorU. The possible use of morpholinonucleoside triphosphates as selective inhibitors of HIV-1 reverse transcriptase is discussed.