DNA Repair Protein XRCC1 Stimulates Activity of DNA Polymerase λ under Conditions of Microphase Separation.

Non-membrane compartments or biomolecular condensates play an important role in the regulation of cellular processes including DNA repair. Here, an ability of XRCC1, a scaffold protein involved in DNA base excision repair (BER) and single-strand break repair, to form protein-rich microphases in the presence of DNA duplexes was discovered. We also showed that the gap-filling activity of BER-related DNA polymerase λ (Pol λ) is significantly increased by the presence of XRCC1.

The XPA Protein-Life under Precise Control.

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process.

Structural features of DNA polymerases β and λ in complex with benzo[a]pyrene-adducted DNA cause a difference in lesion tolerance.

DNA polymerases β (Pol β) and λ (Pol λ) belong to one structural family (X family) and possess the same enzymatic activities. Nonetheless, these enzymes have differences in their catalytic efficiency and specificity. We have previously reported that these enzymes can bypass bulky benzo[a]pyrene-DNA adducts via translesion synthesis during gap-filling reactions, although efficiency and specificity are dependent on the reaction conditions and adduct conformation.

Nucleotide Excision Repair: From Molecular Defects to Neurological Abnormalities.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway, which can remove diverse bulky DNA lesions destabilizing a DNA duplex. NER defects cause several autosomal recessive genetic disorders. Xeroderma pigmentosum (XP) is one of the NER-associated syndromes characterized by low efficiency of the removal of bulky DNA adducts generated by ultraviolet radiation.

Human Tyrosyl-DNA Phosphodiesterase 1 Possesses Transphosphooligonucleotidation Activity With Primary Alcohols.

Human tyrosyl-DNA phosphodiesterase 1 (TDP1) belongs to the phospholipase D superfamily, whose members contain paired catalytic histidine and lysine residues within two conserved motifs and hydrolyze phosphodiester bonds. TDP1 is a DNA repair enzyme that processes 3' DNA end blocking lesions and a wide range of synthetic DNA adducts as a substrate. TDP1 hydrolyzes DNA-adducts via two coordinated S2 nucleophilic attacks mediated by the action of two histidine residues and leads to the formation of the covalent intermediate.

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair.

Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32- and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8-10, 13-22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein.

Replication protein A as a modulator of the poly(ADP-ribose)polymerase 1 activity.

Replication protein A contributes to all major pathways of DNA metabolism and is a target for post-translation modifications, including poly(ADP-ribosyl)ation catalyzed by PARP1. Here we demonstrate that the efficiency of RPA poly(ADP-ribosyl)ation strongly depends on the structure of DNA used for PARP1 activation and on the polarity of RPA binding. Moreover, RPA influences PARP1 activity, and this effect also depends on DNA structure: RPA inhibits PAR synthesis catalyzed by PARP1 in the presence of ssDNA and stimulates it in the presence of a DNA duplex, in particular that containing a nick or a gap.

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure.

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes.

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5'-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5' direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3' direction were hydrolyzed significantly less efficiently.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1.

Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay.

Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position.

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition.

Human DNA polymerases catalyze lesion bypass across benzo[a]pyrene-derived DNA adduct clustered with an abasic site.

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residue in the certain position were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct.

Human and yeast DNA damage recognition complexes bind with high affinity DNA structures mimicking in size transcription bubble.

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. In this study, two types of DNA binding assays were used for the detailed analysis of interaction of these proteins with damaged DNA. An electrophoretic mobility shift assay revealed that human and yeast orthologs behave similarly in DNA binding.

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs.

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates.

Comparative analysis of interaction of human and yeast DNA damage recognition complexes with damaged DNA in nucleotide excision repair.

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding.

Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site.

Human DNA polymerase λ catalyzes lesion bypass across benzo[a]pyrene-derived DNA adduct during base excision repair.

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residues in certain positions were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct.

AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

APE-independent base excision repair (BER) pathway plays an important role in the regulation of DNA repair mechanisms. In this study it has been found that recently discovered tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the AP site cleavage reaction to generate breaks with the 3'- and 5'-phosphate termini. The removal of the 3'-phosphate is performed by polynucleotide kinase phosphatase (PNKP).

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair.

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA-protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5'-side from a lesion.

Photo-cross-linking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct.

Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate.