Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI ...and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3′-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3′-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5′-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.
•Mismatch identity and position in nicked DNA distinctly impact LigI/LigIIIα fidelity.•LigIIIα exceeds LigI in the accuracy of processing short-patch BER DNA intermediates.•Ligation of flapped long-patch BER intermediates results in potential mutagenesis.•The yield of abortive ligation of either substrate depends on its sequence context.
Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and ...accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.
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.
Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are DNA-dependent poly(ADP-ribose)transferases localized in nucleus. They have a significant homology in the C-terminal catalytic domain structure but ...differ in their N-terminal DNA-binding parts. The structural difference has an impact on the interaction of PARP1 and PARP2 with DNA and their DNA-dependent activation. Here, we compare the interaction of PARP1 and PARP2 with free 147 bp nucleosomal DNA and its nucleosome-associated variant (NCP) that contain in one strand a 1-nucleotide gap with 5'-dRP (imitating the intermediate of Base Excision Repair) or no specific damage. The affinity of PARP2 for the DNA strongly depends on the gap presence and to a lesser extent on the association with nucleosomes, while PARP1 interacts primarily with blunt ends of all DNAs and with a lower affinity with the single-strand break. The activities of PARP1 and PARP2 in the autoPARylation reaction and heteromodification of histones are distinctly stimulated by HPF1, depending on the gap presence in activating DNA. The most significant HPF1-induced stimulation of the histone modification in the presence of gapped NCP is a peculiar feature of PARP2. We propose a specific regulatory role of PARP2 in the process of DNA repair in the context of chromatin.
•The activities of PARP1 and PARP2 are distinctly stimulated by HPF1.•The stimulatory action of HPF1 on PARP1/PARP2 automodification depends on the DNA gap presence.•The most significant stimulation of histone modification in the presence of gapped NCP is specific for PARP2.
Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and ...accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg2+ coordinated bonds with PAR polymers.
•Most proteins involved in BER self-associate via homodimerization.•Complexes of PARP1 with various BER proteins are heterotetramers.•The oligomerization state of XRCC1 heterocomplexes depends on the partner.•PARP1 forms huge conglomerates upon the autoPARylation stabilized by magnesium ions.
Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals involved in base excision DNA repair (BER), regulation of gene expression and RNA metabolism. Its major ...enzymatic function is incision of AP sites. Poly(ADP-ribose) polymerase 1 (PARP1) modifies itself and target proteins with poly(ADP-ribose) (PAR), contributing to regulation of many processes. To understand molecular basis of functional cooperation between APE1 and PARP1 in BER, we examined PAR-binding activity and ADP-ribosylation of human APE1 in comparison with known targets of PARP1, using the full-length, N-terminally truncated and catalytically inactive forms of APE1. The protein binds preferentially large ADP-ribose polymers, being very similar to DNA polymerase β (Polβ) but contrasting with the scaffold XRCC1 protein. The interaction with PAR involves the universally conserved catalytic portion and the eukaryote-specific extension of APE1. The ADP-ribosylation of APE1 depends on the structure of PARP1-activating DNA, contrasting APE1 with Polβ and XRCC1. Relative levels of APE1 modification in the presence of different DNA substrates were found to correlate with affinities of the DNAs for APE1 and substrate activities in the enzymatic incision, suggesting the ADP-ribosylation to occur within the DNA-mediated ternary complex. This conclusion was confirmed by importance of the length of DNA region 3’ to the AP site for the modification. Deletion of the N-terminal extension of APE1 produced no significant influence on both the ADP-ribosylation efficiency and hydrolytic stability of the modified protein, suggesting localization of target amino acids in the conserved catalytic portion. The most efficient ADP-ribosylation of the catalytically inactive APE1 mutant was shown to reduce the level of PARP1 automodification, suggesting possible role of APE1 in modulating PARP1 activity. Our data on primary role of DNA in controlling the PARP-catalysed modification provide new insights into mechanisms of protein targeting for ADP-ribosylation.
•Human APE1 is a novel target of PARP1-catalysed modification in vitro.•APE1 interacts with poly(ADP-ribose) using conserved and eukaryote-specific regions.•Targeting of APE1 for ADP-ribosylation is controlled by the interaction with DNA.•The ADP-ribosylation sites of APE1 are localized in the conserved catalytic portion.•The unusual mechanism of ADP-ribosylation may regulate repair of complex DNA lesions.
Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion). Here, ...we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.
Poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerases (PARPs) is one of the immediate cellular responses to DNA damage. The histone PARylation factor 1 (HPF1) discovered recently to form a ...joint active site with PARP1 and PARP2 was shown to limit the PARylation activity of PARPs and stimulate their NAD
-hydrolase activity. Here we demonstrate that HPF1 can stimulate the DNA-dependent and DNA-independent autoPARylation of PARP1 and PARP2 as well as the heteroPARylation of histones in the complex with nucleosome. The stimulatory action is detected in a defined range of HPF1 and NAD
concentrations at which no HPF1-dependent enhancement in the hydrolytic NAD
consumption occurs. PARP2, comparing with PARP1, is more efficiently stimulated by HPF1 in the autoPARylation reaction and is more active in the heteroPARylation of histones than in the automodification, suggesting a specific role of PARP2 in the ADP-ribosylation-dependent modulation of chromatin structure. Possible role of the dual function of HPF1 in the maintaining PARP activity is discussed.
Base excision repair (BER) is a flagship DNA repair system responsible for maintaining genome integrity. Apart from basal enzymes, this system involves several accessory factors essential for ...coordination and regulation of DNA processing during substrate channeling. Y-box-binding protein 1 (YB-1), a multifunctional factor that can interact with DNA, RNA, poly(ADP-ribose) and plenty of proteins including DNA repair enzymes, is increasingly considered as a non-canonical protein of BER. Here we provide quantitative characterization of YB-1 physical interactions with key BER factors such as PARP1, PARP2, APE1, NEIL1 and pol β and comparison of the full-length YB-1 and its C-terminally truncated nuclear form in regard to their binding affinities for BER proteins. Data on functional interactions reveal strong stimulation of PARP1 autopoly(ADP-ribosyl)ation and inhibition of poly(ADP-ribose) degradation by PARG in the presence of YB-1. Moreover, YB-1 is shown to stimulate AP lyase activity of NEIL1 and to inhibit dRP lyase activity of pol β on model DNA duplex structure. We also demonstrate for the first time YB-1 poly(ADP-ribosyl)ation in the presence of RNA.
•YB-1 and its nuclear form physically interact with key BER factors.•YB-1 interaction with PARP1 DNA-binding domain is modulated by DNA.•Poly(ADP-ribosyl)ation of YB-1 can be activated by RNA.•YB-1 is able to stimulate PARP1 activity and to increase the life span of PAR polymer.•YB-1 can modulate NEIL1 and Pol β lyase activities.
In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP ...endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.
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