N. A. Lebedeva

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase β and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase β (pol β), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol β, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol β, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol β may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol β. This enables pol β to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.

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). Tdp1 is known to interact stably with BER proteins: DNA polymerase beta (Pol β), XRCC1, PARP1 and DNA ligase III. The data suggest a role of Tdp1 in the new APE‐independent BER pathway in mammals.

DNA Polymerases β and λ Bypass Thymine Glycol in Gapped DNA Structures

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

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. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

Trapping of Human DNA Topoisomerase I by DNA Structures Mimicking Intermediates of DNA Repair

In this report we show that human DNA Topoisomerase I (Top1) forms DNA‐protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products were identified by SDS‐gel electrophoresis. The chemical structure of the groups at 5′ or 3′ end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and other nick‐sensors. Top1‐DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability.

Sensitized photomodification of mammalian DNA polymerase β. A new approach for highly selective affinity labeling of polymerases

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base‐substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site‐bound photosensitizer for photoaffinity modification of DNA polymerase β (pol β). 5′‐[32P]‐labeled primer was elongated in situ by pol β with a photoreactive analog of TTP (FAB‐4‐dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365–450 nm), can activate the photoreagent, crosslinking it to pol β as a result of fluorescence resonance energy transfer. The initial rate of pol β photomodification was shown to increase by a factor of ten. The selectivity of pol β photosensitized modification was proved by adding human replication protein A.

Interaction between DNA polymerase λ and RPA during translesion synthesis

Replication of damaged DNA (translesion synthesis, TLS) is realized by specialized DNA polymerases. Additional protein factors such as replication protein A (RPA) play important roles in this process. However, details of the interaction are unknown. Here we analyzed the influence of the hRPA and its mutant hABCD lacking domains responsible for protein-protein interactions on ability of DNA polymerase λ to catalyze TLS. The primer-template structures containing varying parts of extended strand (16 and 37 nt) were used as model systems imitating DNA intermediate of first stage of TLS. The 8-oxoguanine disposed in +1 position of the template strand in relation to 3′-end of primer was exploited as damage. It was shown that RPA stimulated TLS DNA synthesis catalyzed by DNA polymerase λ in its globular but not in extended conformation. Moreover, this effect is dependent on the presence of p70N and p32C domains in RPA molecule.

DNA polymerases β and λ as potential participants of TLS during genomic DNA replication on the lagging strand

The main strategy used by pro-and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases β and λ on DNA substrates using mono-or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg2+ or Mn2+. DNA polymerases β and λ were able to catalyze DNA synthesis across THF only in the presence of Mn2+. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase β. As for DNA polymerase λ, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase β. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase λ. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases β and λ on DNA templates containing damage.

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. Digestion with uracil DNA glycosylase was utilized to generate an AP site which was then hydrolyzed by APE1, and the resulting gap was processed by X-family DNA polymerases β (Polβ) and λ (Polλ), or Y-family polymerase ι (Polι). By varying reaction conditions, namely, Mg2+/Mn2+ replacement/combination and ionic strength decrease, we found that under certain conditions both Polβ and Polι can catalyze lesion bypass across both cis- and trans-BP adducts in the presence of physiological dNTP concentrations. Polβ and Polι catalyze gap filling trans-lesion synthesis in an error prone manner. By contrast, Polλ selectively introduced the correct dCTP opposite the modified dG in the case of cis-BP-dG adduct only, and did not bypass the stereoisomeric trans-adduct under any of the conditions examined. The results suggest that Polλ is a specialized polymerase that can process these kinds of lesions.