Human cells respond to DNA damage with an acute and transient burst in production of poly(ADP-ribose), a posttranslational modification that expedites damage repair and plays a pivotal role in cell ...fate decisions. Poly(ADP-ribose) polymerases (PARPs) and glycohydrolase (PARG) are the key set of enzymes that orchestrate the rise and fall in cellular levels of poly(ADP-ribose). In this perspective, we focus on recent structural and mechanistic insights into the enzymes involved in poly(ADP-ribose) production and turnover, and we highlight important questions that remain to be answered.
DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in ...all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.
DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on
Xenopus egg extracts ...that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase ζ. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports ...obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.
Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75r which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular ...binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of α-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.
Tomkinson et al describe the structural and mechanistic insights provided by crystallographic studies of DNA ligases. Protein-protien interactions may coordinate the final ligation step with the ...earlier steps of DNA metabolic pathways.
The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The ...PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.
Background: The DNA repair scaffold XRCC1 binds to poly(ADP-ribose)ylated PARP1 at damaged chromatin.
Results: XRCC1 preferentially binds to poly(ADP-ribose) chains longer than 7 ADP-ribose units in length.
Conclusion: We identify specific determinants of XRCC1-PARP1 complex assembly, and disassembly by PARG.
Significance: Our TR-FRET assay is useful for investigating turnover of posttranslational modifications and for identifying inhibitors by high-throughput screening.
The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized ...mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase ...bypasses the common oxidative lesion 8‐oxo‐7, 8‐dihydro‐2′‐deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn)·dA (anti) in a Hoogsteen‐like base pair at the 3′ terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG·dA mismatch, thus providing insight into the high mutagenic potential of 8oG.
Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated ...how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (kst) and spontaneous N-glycosidic bond hydrolysis (knon) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (kst/knon = 108), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.
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