Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Background: Topoisomerase V is a unique enzyme and the sole member of the IC subtype of type I topoisomerases. Results: We probed the role of several amino acids comprising the topoisomerase active site to elucidate their role in catalysis. Conclusion:The work reveals the role of several amino acids and suggests interesting parallels with type IB topoisomerases. Significance: The results expand our understanding of this new subtype of topoisomerases.

Section: Discussionmentioning

confidence: 72%

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confidence: 99%

Biochemical Characterization of the Topoisomerase Domain of Methanopyrus kandleri Topoisomerase V

Rajan

Osterman

Gast

et al. 2014

“…The individual catalytic residues Arg-130, Lys-167, Arg-223, and His-265 accelerate the chemical step by factors of 10 5 , 10 4 , 10 5 , and 10 2 , respectively (13)(14)(15). Lys-167 and Arg-130 comprise a proton relay that catalyzes the expulsion of the 5Ј-O of the leaving DNA strand (19,21). His-265 donates a hydrogen bond to the nonbridging pro-Sp oxygen of the scissile phosphodiester to stabilize the transition state (16,56).…”

Section: Resultsmentioning

confidence: 99%

“…The active sites of poxvirus and nuclear type IB topoisomerases consist of five conserved functional groups, e.g. Arg-130, Lys-167, Arg-220, His-265, and Tyr-274 in vaccinia virus topoisomerase IB (vaccinia TopIB), which execute the cleavage and religation transesterification steps of the catalytic cycle (13)(14)(15)(16)(17)(18)(19)(20)(21).…”

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confidence: 99%

Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Patel

Shuman

Mondragón

2006

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Topoisomerases I and II are involved in virtually all DNA transactions and are the targets of clinically effective anti-cancer and anti-infective drugs (1, 2). Topoisomerases exploit a tyrosine nucleophile to attack the phosphodiester backbone, yielding a covalent enzyme-DNA adduct on one side of the resulting break that permits the passage of strand(s) through the break. Type I enzymes operate by cleaving one DNA strand and passing another strand through the nick; type II enzymes cleave both DNA strands and allow passage of a duplex segment through the double strand break. Closure of the break by reversal of the cleavage step results in a change to the topology of DNA, with no net effect on its chemical structure.Type I topoisomerases are subclassified as type IA or type IB enzymes, depending on whether they form a 5Ј-or 3Ј-phosphotyrosyl adduct, respectively. Type IA enzymes are distributed widely in the bacterial, archaeal, and eukaryal domains of life. Type IB enzymes are found in eukarya, eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria (1-6). Eukaryotic nuclear type IB enzymes are large monomeric polypeptides, typically Ͼ90 kDa, whereas the viral and bacterial type IB polypeptides are much smaller, typically ϳ33-36 kDa. Despite their differences in size, the poxvirus and nuclear type IB enzymes have a common core tertiary structure and catalytic mechanism (7,8), which is shared with the tyrosine recombinase family (9 -12), thereby suggesting a common ancestry for type IB topoisomerases and tyrosine recombinases (7,8). The active sites of poxvirus and nuclear type IB topoisomerases consist of five conserved functional groups, e.g. Arg-130, Lys-167, Arg-220, His-265, and Tyr-274 in vaccinia virus topoisomerase IB (vaccinia TopIB), which execute the cleavage and religation transesterification steps of the catalytic cycle (13-21).Vaccinia TopIB is composed of two domains separated by a flexible protease-sensitive linker (22, 23). The 234-aa 3 carboxyl-terminal (C) domain contains the active site and catalyzes DNA transesterification and supercoil relaxation but has reduced affinity for DNA compared with the full-length enzyme (24). The 80-aa amino-terminal (N) domain interacts with DNA in the major groove (22,25,26). The atomic structures of the individual domains of vaccinia TopIB have been determined by x-ray crystallography (7,22). Whereas the fold and active site of the predominantly ␣-helical catalytic domain are conserved in nuclear type IB topoisomerases (nuclear TopIB) and the tyrosine recombinases (7), the amino-terminal domain, which consists primarily of ␤-strands (22), has no counterpart in tyrosine recombinases, although it is structurally homologous to part of the much larger amino-terminal domain of nuclear TopIB (27).Bacterial type IB topoisomerases were discovered recently (5) and are similar to poxvirus type IB topoisomerases with respect to their size, primary structures, and domain organization (Fig. 1). Mutational analyses indicate that the transesterification mec...

“…(The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2)(3)(4)(5)(6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the CCCTT element (7)(8)(9)(10)(11)(12).…”

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confidence: 99%

Nonpolar Nucleobase Analogs Illuminate Requirements for Site-specific DNA Cleavage by Vaccinia Topoisomerase

Yakovleva

Lai

Kool

et al. 2006

Self Cite

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 nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the ؉2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the ؉1T base was not elided by F substitution. We impute a role for ؉1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the ؉4G and ؉3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for ؉4G and ؉3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the ؉1A base of the nonscissile strand; the reaction equilibrium is restored by ؉1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner.Vaccinia virus DNA topoisomerase IB cleaves and rejoins one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate formed at a specific pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (1). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2-6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the C...