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. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase ß-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.

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.

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. 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 ß (Polß) 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 Polß 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.

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 ß (Polß), 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.

Novel Inhibitors of DNA Repair Enzyme TDP1 Combining Monoterpenoid and Adamantane Fragments.

BACKGROUND AND OBJECTIVE: The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors. METHODS: New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling. RESULTS: 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549. CONCLUSION: The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect.

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. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.

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 ß (Polß), 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-Polß, APE1-TDP1, APE1-PARP1 and Polß-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 Polß. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polß complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polß, 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 Polß 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.

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. 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.

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. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.