Summary Type IB DNA topoisomerases (TopIB) are enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and, in the case of poxvirus TopIB, form protein-DNA synaptic filaments. Here we report a structure of D. radiodurans TopIB in complex with a 12-bp duplex DNA that demonstrates a secondary DNA binding site located on the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. In support of this, mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr 274 were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH 3 and NO 2 groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5- interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120-and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120-and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.Type IB DNA topoisomerases (TopIB) 2 relax DNA supercoils by repeatedly breaking and rejoining one strand of the DNA duplex through a covalent DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia virus TopIB, which displays stringent specificity for cleavage at the sequence 5Ј(C/T)CCTT2 in the scissile strand (1-5), has been an instructive model system for mechanistic studies of the TopIB family. Analyses of the effects of vaccinia TopIB mutations and DNA target site modifications revealed that the cleavage and religation transesterification reactions are driven by four conserved amino acid side chains (Arg 130 , Lys 167 , Arg 223 , and His 265 ) that compose the active site and catalyze the attack of Tyr 274 on the scissile phosphodiester (6 -10). The two arginines and the histidine interact directly with the scissile phosphodiester ( Fig. 1) and are proposed to stabilize the developing negative charge on a putative pentacoordinate phosphorane transition state (11-15). Lys 167 serves together with Arg 220 as a general acid to expel the 5Ј-OH leaving group during the cleavage reaction (16,17). One of the outstanding problems in TopIB biochemistry concerns whether and how the enzyme might activate the tyrosine nucleophile via general base catalysis.The poxvirus TopIB active site is not preassembled prior to DNA binding. The apoenzyme crystal structure of vaccinia TopIB showed that three of the catalytic residues (Arg 130 , Lys 167 , and Tyr 274) are either disordered or out of position to perform transesterification chemistry (18). Tyr 274 in the apoenzyme is located far away from the scissile phosphate and the other catalytic amino acids (Fig. 2A). The act of DNA binding elicits a major rearrangement of Tyr 274 and the surrounding ␣-helix that brings Tyr 274 into the active site in proximity to Arg 223 . The conformational switch occurs by melting a long ␣-heli...

DNA ligase D (LigD) participates in a mutagenic pathway of nonhomologous end joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase module. The POL domain performs templated and nontemplated primer extension reactions with either dNTP or rNTP substrates. Here we report that Pseudomonas LigD POL is an unfaithful nucleic acid polymerase. Although the degree of infidelity in nucleotide incorporation varies according to the mispair produced, we find that a correctly paired ribonucleotide is added to the DNA primer terminus more rapidly than the corresponding correct deoxyribonucleotide and incorrect nucleotides are added much more rapidly with rNTP substrates than with dNTPs, no matter what the mispair configuration. We find that 3 mispairs are extended by LigD POL, albeit more slowly than 3 paired primer-templates. The magnitude of the rate effect on mismatch extension varies with the identity of the 3 mispair, but it was generally the case that mispaired ends were extended more rapidly with rNTP substrates than with dNTPs. These results lend credence to the suggestion that LigD POL might fill in short 5-overhangs with ribonucleotides when repairing double strand breaks in quiescent cells. We report that LigD POL can add a deoxynucleotide opposite an abasic lesion in the template strand, albeit slowly. Ribonucleotides are inserted more rapidly at an abasic lesion than are deoxys. LigD POL displays feeble activity in extending a preformed primer terminus opposing an abasic site, but can readily bypass the lesion by slippage of the primer 3 di-or trinucleotide and realignment to the template sequence distal to the abasic site. Covalent benzo[a]pyrene-dG and benzo[c]phenanthrene-dA adducts in the template strand are durable roadblocks to POL elongation. POL can slowly insert a dNMP opposite the adduct, but is impaired in the subsequent extension step. Bacterial DNA ligase D (LigD)2 is distinguished from all other DNA ligases insofar as its enzymatic activity is not limited to sealing DNA strands. Rather, it is a polyfunctional repair enzyme consisting of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase domain (1-10). Interest in LigD is driven by evidence that it collaborates with a bacterial Ku homolog in a nonhomologous end joining (NHEJ) pathway characterized by a high incidence of frameshift mutations at the sites of double strand break (DSB) repair (1-4, 9, 10). Biochemical characterization of the POL and phosphoesterase components suggests that they provide a means of remodeling the 3Ј ends of broken DNA strands prior to sealing by the ligase component (2-10).The LigD POL domain catalyzes either nontemplated single nucleotide additions to a blunt-ended duplex DNA or fill-in synthesis at a 5Ј-tailed duplex DNA; these are the molecular signatures of mutagenic mycobacterial NHEJ in vivo at bluntend and 5Ј-overhang DSBs, respectively (2, 9, 10). POL activity in vitro is optimal in the presence of manganese (...

Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3′ to 5′ on single-stranded DNA and has 3′ to 5′ helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N 2 -deoxyguanosine and N 6 -deoxyadenosine positions within the 3-sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the ؉6 and ؉5 base pairs, slowed 4-fold by intercalation between the ؉5 and ؉4 base pairs, and virtually abolished by BcPh intercalation between the ؉4 and ؉3 base pairs and the ؉3 and ؉2 base pairs. Intercalation between the ؉2 and ؉1 base pairs by the ؉2R BcPh dA adduct abolished transesterification, whereas the overlapping ؉1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the ؉1 and ؊1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the ؊1 and ؊2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the ؊2 and ؊3 base pairs had little effect. The anthracycline drug nogalamycin, a noncovalent intercalator with preference for 5-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC 50 of 0.7 M. Nogalamycin was most effective when the drug was preincubated with DNA and when the cleavage target site was 5-CCCTT2G instead of 5-CCCTT2A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.Poxvirus DNA topoisomerase I is important for virus replication (1) and a potential target for drug therapy of smallpox, in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared with human topoisomerase I (2-6). Poxvirus topoisomerases are exemplary type IB family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (3). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (6 -10). Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the DNA target (11,12).Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (13)(14)(15)(16)(17)(18)(19). Re...