Hierarchy of lesion processing governs the repair, double-strand break formation and mutability of three-lesion clustered DNA damage

Ionising radiation induces clustered DNA damage sites which pose a severe challenge to the cell’s repair machinery, particularly base excision repair. To date, most studies have focussed on two-lesion clusters. We have designed synthetic oligonucleotides to give a variety of three-lesion clusters containing abasic sites and 8-oxo-7, 8-dihydroguanine to investigate if the hierarchy of lesion processing dictates whether the cluster is cytotoxic or mutagenic. Clusters containing two tandem 8-oxoG lesions opposing an AP site showed retardation of repair of the AP site with nuclear extract and an elevated mutation frequency after transformation into wild-type or mutY Escherichia coli. Clusters containing bistranded AP sites with a vicinal 8-oxoG form DSBs with nuclear extract, as confirmed in vivo by transformation into wild-type E. coli. Using ung1 E. coli, we propose that DSBs arise via lesion processing rather than stalled replication in cycling cells. This study provides evidence that it is not only the prompt formation of DSBs that has implications on cell survival but also the conversion of non-DSB clusters into DSBs during processing and attempted repair. The inaccurate repair of such clusters has biological significance due to the ultimate risk of tumourigenesis or as potential cytotoxic lesions in tumour cells

Direct visualization of joining of DNA fragments by LigIIIβ using atomic force microscopy

DNA ligaseIII is one of three mammalian DNA ligases (LigI, LigIII and LigIV). LigIII is distinguished from the other ligases by the presence of a Zinc finger(ZnF) that improves ligation efficiency. Previously, it was demonstrated that LigIII has two different DNA binding modules (ZnF-DBD and NTase-OB) and a jack-knife model has been proposed to explain nick recognition and joining of double-strand breaks. However, the oligomeric state of LigIII in solution and during ligation, and the role of ZnF in end-end ligation are unknown. Using atomic force microscopy, we directly visualized a germ cell-specific form of DNA LigIII, LigIIIb and a delZnF mutant, their interactions with DNA and ligation products. We found no evidence for oligomerization of WT and delZnF LigIIIb in solution or when complexed to DNA. Importantly, WT and delZnF proteins exist in three distinct conformational states: closed, semi-extended, and extended conformations. While WTLigIIIb protein accesses all three conformational states significantly, delZnF LigIIIb occupies primarily the closed and semi-extended states, suggesting that ZnF is part of one wing as proposed in the jack-knife model. Furthermore, binding and ligation studies on nicked and non-nicked blunt-end DNA and DNA with 5\u27 overhang indicate that in addition to tandem joining of two linear DNA molecules, LigIIIb can mediate the ligation of a variety of higher order structures including three way junctions. In addition, with 5\u27 overhang DNA, compared to WT protein, delZnF promotes a small but significant occurrence of three-way junctions, lassoes and knots in the presence of MgCl2. These data suggest that monomeric DNA LigIIIb is capable of binding two DNA molecules simultaneously. Currently, we are testing the hypothesis that the ZnF in LigIIIb may be involved in quality control ensuring only tandem end-end ligation

Kinetic Characterization of Single Strand Break Ligation in Duplex DNA by T4 DNA Ligase

Background: T4 DNA ligase catalyzes the formation of phosphodiester bonds in dsDNA

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase*

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps

Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase*

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3′-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3′-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3′-OH nucleoside in the catalysis of DNA 5′-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig

Molecular Mechanism of DNA Deadenylation by the Neurological Disease Protein Aprataxin*

The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5′-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5′ terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA