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).

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair.

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.

The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate.

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.

Dual function of HPF1 in the modulation of PARP1 and PARP2 activities.

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.

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA.

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.

Human apurinic/apyrimidinic endonuclease 1 is modified in vitro by poly(ADP-ribose) polymerase 1 under control of the structure of damaged DNA.

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.

Dynamic light scattering study of base excision DNA repair proteins and their complexes.

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.

Y-box-binding protein 1 as a non-canonical factor of base excision repair.

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.

Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions.

Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

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.

Clustered DNA lesions containing 5-formyluracil and AP site: repair via the BER system.

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.

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step.

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis.