A nick-sensing DNA 3'-repair enzyme from Arabidopsis

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3'-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3'-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys(3)-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by approximately 73 degrees upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in the Arabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3'-phosphoesterase acts as a "nick sensor" for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex.     

The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA

The recognition of double-stranded DNA breaks and single-stranded nicks by human poly(ADP-ribose) polymerase and the consequent enzymic activation were examined using derivatives of the enzyme expressed in Escherichia coli. The N-terminal 162 residues encompass two zinc fingers. Deletion or mutation of the first finger results in a loss of activation by DNA with either single-stranded or double-stranded damage. Destruction of the second finger reduces activation by double-stranded DNA breaks only slightly, but eliminates activation by single-stranded DNA nicks. These data suggest that activation by single-stranded DNA nicks requires two zinc fingers, but activation by double-stranded DNA breaks requires only the finger closer to the N terminus. Variant proteins that lack both zinc fingers are enzymically inactive but still exhibit weak DNA binding, which is independent of DNA damage. Thus, other regions are also capable of binding intact DNA, but the recognition of a strand nick or break which occasions the synthesis of poly(ADP-ribose) specifically requires the zinc fingers.     

Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FokI. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by ~4 bp with a 5′ overhang, as for native FokI. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

Structural recognition of DNA by poly(ADP-ribose)polymerase-like zinc finger families

PARP-like zinc fingers (zf-PARPs) are protein domains apt to the recognition of multiple DNA secondary structures. They were initially described as the DNA-binding, nick-sensor domains of poly(ADP-ribose)polymerases (PARPs). It now appears that zf-PARPs are evolutionary conserved in the eukaryotic lineage and associated with various enzymes implicated in nucleic acid transactions. In the present study, we discuss the functional and structural data of zf-PARPSs in the light of a comparative analysis of the protein family. Sequence and structural analyses allow the definition of the conserved features of the zf-PARP domain and the identification of five distinct phylogenetic groups. Differences among the groups accumulate on the putative DNA binding surface of the PARP zinc-finger fold. These observations suggest that different zf-PARP types have distinctive recognition properties for DNA secondary structures. A comparison of various functional studies confirms that the different finger types can accomplish a selective recognition of DNA structures.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Role of base flipping in specific recognition of damaged DNA by repair enzymes

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G.U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol(-1) lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol(-1) lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol(-1), making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.     

Influence of a cis,syn‐cyclobutane pyrimidine dimer damage on DNA conformation studied by molecular dynamics simulations

The photo‐induced formation of cis‐syn‐cyclobutane pyrimidine dimers (CPD) is a highly mutagenic and cancerogenic DNA lesion. In bacteria photolyases can efficiently reverse the dimer formation employing a light‐driven reaction after looping out the CPD damaged bases into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The CPD damage may alter the DNA structure and dynamics already in the absence of the repair enzyme which can facilitate the initial binding of a photolyase repair enzyme. To characterize the effect of a CPD damage, extensive comparative molecular dynamics (MD) simulations on duplex DNA with central regular or CPD damaged nucleotides were performed supplemented with simulations of the DNA‐photolyase complex. Although no spontaneous flipping out transitions of the damaged bases were observed, the simulations showed significant differences in the conformational states of regular and CPD damage DNA. The isolated damaged DNA adopted transient conformations which resembled the global shape of the repair enzyme bound conformation more closely compared to regular B‐DNA. In particular, these conformational changes were observed in most of helical and structural parameters where the protein bound DNA differs drastically from regular B‐DNA. It is likely that the transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out process of the damaged DNA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 215–222, 2015.     

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.     

Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism

Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA. A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli. The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches. Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product. This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA. When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition. The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases. Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C. A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.     

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.