14 research outputs found
MECHANISTIC INSIGHT INTO THE ROLE OF POLY(ADP-RIBOSYL)ATION IN DNA TOPOLOGY MODULATION AND RESPONSE TO DNA DAMAGE
Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by
reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand
breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability
and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further
disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse
the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor
molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand
breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly
branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis
may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR
corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand
break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that
this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology
modulation would enable nucleosome displacement and chromatin decondensation around the
lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated
DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order
chromatin structures. The available structures of PARP1 are consistent with the strand break induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis
Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation
DNA-dependent poly(ADP-ribose) polymerases (PARPs) PARP1, PARP2 and PARP3 act as DNA break sensors signalling DNA damage. Upon detecting DNA damage, these PARPs use nicotine adenine dinucleotide as a substrate to synthesise a monomer or polymer of ADP-ribose (MAR or PAR, respectively) covalently attached to the acceptor residue of target proteins. Recently, it was demonstrated that PARP1–3 proteins can directly ADP-ribosylate DNA breaks by attaching MAR and PAR moieties to terminal phosphates. Nevertheless, little is still known about the mechanisms governing substrate recognition and specificity of PARP1, which accounts for most of cellular PARylation activity. Here, we characterised PARP1-mediated DNA PARylation of DNA duplexes containing various types of breaks at different positions. The 3′-terminal phosphate residue at double-strand DNA break ends served as a major acceptor site for PARP1-catalysed PARylation depending on the orientation and distance between DNA strand breaks in a single DNA molecule. A preference for ADP-ribosylation of DNA molecules containing 3′-terminal phosphate over PARP1 auto-ADP-ribosylation was observed, and a model of DNA modification by PARP1 was proposed. Similar results were obtained with purified recombinant PARP1 and HeLa cell-free extracts. Thus, the biological effects of PARP-mediated ADP-ribosylation may strongly depend on the configuration of complex DNA strand breaks
IS THERE AN ADVANTAGEOUS ARRANGEMENT OF AROMATIC RESIDUES IN PROTEINS? STATISTICAL ANALYSIS OF AROMATIC INTERACTIONS IN GLOBULAR PROTEINS
The aim of this study was to evaluate the favorability of different conformations of aromatic residues in
proteins by analysing the occurrence of particular conformations. The clustering of protein structures
from the Protein Data Bank (PDB) was performed. Conformations of interacting aromatic residues were
analyzed for 511 282 pairs in 35 493 protein structures sharing less than 50% identity. Pairs with a par allel arrangement of aromatic residues made up 6.2% of all possible ones, which was twice as much as
expected. Pairs with a perpendicular arrangement of aromatic residues made up 25%. We demonstrate
that the most favorable arrangement was at an angle of 60 between the interacting aromatic residues.
Among all possible aromatic pairs, the His-His pair was twice as frequent as expected, and the His-Phe
pair was less frequent than expected. A server (CARP – Contacts of Aromatic Residues in Proteins) has
been created for calculating essential structural features of interacting aromatic residues in protein
DNA-HISTONE CROSS-LINKS: FORMATION AND REPAIR
The nucleosome is a stretch of DNA wrapped around a histone octamer. Electrostatic
interactions and hydrogen bonds between histones and DNA are vital for the stable
organization of nucleosome core particles, and for the folding of chromatin into more
compact structures, which regulate gene expression via controlled access to DNA. As
a drawback of tight association, under genotoxic stress, DNA can accidentally crosslink to histone in a covalent manner, generating a highly toxic DNA-histone cross-link
(DHC). DHC is a bulky lesion that can impede DNA transcription, replication, and
repair, often with lethal consequences. The chemotherapeutic agent cisplatin, as well
as ionizing and ultraviolet irradiations and endogenously occurring reactive aldehydes,
generate DHCs by forming either stable or transient covalent bonds between DNA and
side-chain amino groups of histone lysine residues. The mechanisms of DHC repair
start to unravel, and certain common principles of DNA-protein cross-link (DPC) repair
mechanisms that participate in the removal of cross-linked histones from DNA have
been described. In general, DPC is removed via a two-step repair mechanism. First,
cross-linked proteins are degraded by specific DPC proteases or by the proteasome,
relieving steric hindrance. Second, the remaining DNA-peptide cross-links are eliminated
in various DNA repair pathways. Delineating the molecular mechanisms of DHC repair
would help target specific DNA repair proteins for therapeutic intervention to combat
tumor resistance to chemotherapy and radiotherapy
XPC DEFICIENCY INCREASES RISK OF HEMATOLOGIC MALIGNANCIES THROUGH MUTATOR PHENOTYPE AND CHARACTERISTIC MUTATIONAL SIGNATURE
Recent studies demonstrated a dramatically increased risk of leukemia in patients with a rare
genetic disorder, Xeroderma Pigmentosum group C (XP-C), characterized by constitutive
deficiency of global genome nucleotide excision repair (GG-NER). The genetic mechanisms of
non-skin cancers in XP-C patients remain unexplored. In this study, we analyze a unique
collection of internal XP-C tumor genomes including 6 leukemias and 2 sarcomas. We
observe a specific mutational pattern and an average of 25-fold increase of mutation rates in
XP-C versus sporadic leukemia which we presume leads to its elevated incidence and early
appearance. We describe a strong mutational asymmetry with respect to transcription and
the direction of replication in XP-C tumors suggesting association of mutagenesis with bulky
purine DNA lesions of probably endogenous origin. These findings suggest existence of a
balance between formation and repair of bulky DNA lesions by GG-NER in human body cells
which is disrupted in XP-C patients
Aberrant base excision repair pathway of oxidatively damaged DNA: Implications for degenerative diseases
Abstract In cellular organisms composition of DNA is constrained to only four nucleobases A, G, T and C, except for minor DNA base modifications such as methylation which serves for defence against foreign DNA or gene expression regulation. Interestingly, this severe evolutionary constraint among other things demands DNA repair systems to discriminate between regular and modified bases. DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, the mismatched base pairs in DNA can occur from a spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated BER and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Importantly, in DNA repair deficient cells bacterial MutY, human TDG and mammalian MMR can act in the aberrant manner: MutY and TDG removes adenine and thymine opposite misincorporated 8-oxoguanine and damaged adenine, respectively, whereas MMR removes thymine opposite to O6-methylguanine. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences accumulated showing that in addition to the accumulation of oxidatively damaged DNA in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. For example, the aberrant BER and MMR pathways for oxidized guanine residues can lead to trinucleotide expansion that underlies Huntington's disease, a severe hereditary neurodegenerative syndrome. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidized base modifications and their possible role in age-related diseases
Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases
It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil–DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth’s atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions
Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways
International audienceOxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins
Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.
International audienceOxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichiacoli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg2+ dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases