A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase ß, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins.

Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS-nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS's affinity for a nucleic acid polymer. Some proteins-which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates-disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.

Inhibitory Effects of 7-Methylguanine and Its Metabolite 8-Hydroxy-7-Methylguanine on Human Poly(ADP-Ribose) Polymerase 1.

Previously, we have found that a nucleic acid metabolite, 7-methylguanine (7mGua), produced in the body can have an inhibitory effect on the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, an important pharmacological target in anticancer therapy. In this work, using an original method of analysis of PARP1 activity based on monitoring fluorescence anisotropy, we studied inhibitory properties of 7mGua and its metabolite, 8-hydroxy-7-methylguanine (8h7mGua). Both compounds inhibited PARP1 enzymatic activity in a dose-dependent manner, however, 8h7mGua was shown to be a stronger inhibitor. The IC50 values for 8h7mGua at different concentrations of the NAD+ substrate were found to be 4 times lower, on average, than those for 7mGua. The more efficient binding of 8h7mGua in the PARP1 active site is explained by the presence of an additional hydrogen bond with the Glu988 catalytic residue. Experimental and computational studies did not reveal the effect of 7mGua and 8h7mGua on the activity of other DNA repair enzymes, indicating selectivity of their inhibitory action.

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

DNA polymerases ß (Pol ß) 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. In the present study, we analyzed structural features of Pols ß and λ complexed with a gapped DNA duplex containing either cis- or trans-benzo[a]pyrene-diol epoxide-N2-dG (BP-dG) using molecular dynamics simulations. It was found that the most pronounced structural difference lies in the positioning of the trans-BP-dG residue relative to secondary structures of the protein; this dissimilarity may explain the differences between Pols ß and λ in gap-filling/translesion synthesis. In the case of Pol ß, trans-BP-dG turned out to be positioned parallel to the α-helix and ß-sheet. In the Pol λ complex, trans-BP-dG is perpendicular to the α-helix. This difference persisted throughout the molecular dynamics trajectory. Selectivity for the BP-dG isomers remained after a deletion of noncatalytic domains of Pol λ. Modeling of Pol λ or ß complexes with cis-BP-dG-containing DNA in the presence of Mn2+ either at both metal-binding sites or at the catalytic site only revealed that for both enzymes, the model of the complex containing both Mg2+ and Mn2+ is stabler than that containing two Mn2+ ions. This observation may reflect a shared property of these enzymes: the preference for Mn2+ in terms of catalysis and for Mg2+ regarding triphosphate coordination during the translesion reaction.

Strand Displacement Activity of PrimPol.

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.

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. Using fluorescently labeled proteins, we showed their direct interaction and characterized it quantitatively. RPA can accelerate the replacement of poly(ADP-ribosyl)ated PARP1 molecules bound to DNA by the unmodified ones. Thus, our data allow us to suggest that the balance between the affinities of PARP1 and RPA for DNA and the interaction of these proteins with each other are the cornerstone of the modulating effect of RPA on PARP1 activity. This effect might contribute to the regulation of PARP1 activity in various DNA processing mechanisms including DNA replication and repair pathways, where both PARP1 and RPA participate.

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. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

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. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using (32)P-labeled NAD(+) and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.

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. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed using fluorescent depolarization measurements. The XPC-RAD23B and the Rad4-Rad23 proteins bind to the damaged 15 nt bubble-DNA structure mimicking in size the "transcription bubble" DNA intermediate with the highest affinity (KD values ~10(-10) M or less) that is reduced in the following order: damaged bubble > undamaged bubble > damaged duplex > undamaged duplex. The affinity of XPC/Rad4 for various DNAs was shown to correlate with DNA bending angle. The results obtained show clearly that more deviation from regular DNA structure leads to higher XPC/Rad4 affinity.

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. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (K(D) ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nM, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.

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. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5'-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5'-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages.

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages.

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive "damages" were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase beta using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were performed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg(2+) whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair.