Study of Interaction of the PARP Family DNA-Dependent Proteins with Nucleosomes Containing DNA Intermediates of the Initial Stages of BER Process

Biochemistry (Moscow) - Reaction of (ADP-ribosyl)ation catalyzed by DNA-dependent proteins of the poly(ADP-ribose)polymerase (PARP) family, PARP1, PARP2, and PARP3, comprises the cellular response...     

Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

Use of Modified Flap Structures for Study of Base Excision Repair Proteins

To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.     

Effect of Nucleotide Replacements in tRNAPhe on Positioning of the Acceptor End in the Complex with Phenylalanyl-tRNA Synthetase

The effect of replacement of tRNA(Phe) recognition elements on positioning of the 3'-terminal nucleotide in the complex with phenylalanyl-tRNA synthetase (PheRS) from T. thermophilus in the absence or presence of phenylalanine and/or ATP has been studied by photoaffinity labeling with s(4)U76-substituted analogs of wild type and mutant tRNA(Phe). The double mutation G34C/A35U shows the strongest disorientation in the absence of low-molecular-weight substrates and sharply decreases the protein labeling, which suggests an initiating role of the anticodon in generation of contacts responsible for the acceptor end positioning. Efficiency of photo-crosslinking with the alpha- and beta-subunits in the presence of individual substrates is more sensitive to nucleotide replacements in the anticodon (G34 by A or A36 by C) than to changes in the general structure of tRNA(Phe) (as a result of replacement of the tertiary pair G19-C56 by U19-G56 or of U20 by A). The degree of disorders in the 3'-terminal nucleotide positioning in the presence of both substrates correlates with decrease in the turnover number of aminoacylation due to corresponding mutations. The findings suggest that specific interactions of the enzyme with the anticodon mainly promote the establishment (controlled by phenylalanine) of contacts responsible for binding of the CCA-end and terminal nucleotide in the productive complex, and the general conformation of tRNA(Phe) determines, first of all, the acceptor stem positioning (controlled by ATP). The main recognition elements of tRNA(Phe), which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm.     

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.     

Human DNA polymerase λ catalyzes lesion bypass across benzo[a]pyrene-derived DNA adduct during base excision repair

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 residues in certain positions were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. The resulting DNA duplexes contained dU either directly opposite the modified dG or shifted to adjacent 5' (-1) or 3' (+1) positions. Digestion with uracil DNA glycosylase was utilized to generate AP sites which were then hydrolyzed by APE1, and the resulting gaps were processed by DNA polymerase β (Polβ) or λ (Polλ). The AP sites in position -1 can be repaired effectively using APE1 and Polβ or Polλ. The AP sites opposite the BP lesions can be repaired using Polλ in the case of cis- but not the trans-isomeric adduct. The AP sites in position +1 are the most difficult to repair. In the case of the AP site located in position +1, the activity of Polλ does not depend on the stereoisomeric properties of the BP lesions and dCTP is the preferred inserted substrate in both cases. The capability of Polλ to introduce the correct dNTP opposite the cis-BP-dG adduct in gap filling reactions suggests that this polymerase may play a specialized role in the process of repair of these kinds of lesions.     

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 ?. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.