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Discovering the role of ATP hydrolysis in the catalytic cycle of type II Topoisomerases

Discovering the role of ATP hydrolysis in the catalytic cycle of type II Topoisomerases

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Published by Cassidy Crook

A comprehensive review of type II Topoisomerases and elucidation of the basis for coupling the free energy of ATP hydrolysis to the exergonic reaction of DNA relaxation.

A comprehensive review of type II Topoisomerases and elucidation of the basis for coupling the free energy of ATP hydrolysis to the exergonic reaction of DNA relaxation.

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Published by: Cassidy Crook on Jan 29, 2013
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Mechanistic Properties of Type II Topoisomerases: The Role of ATP Hydrolysis
Cassidy Crook 
 Within the late 1960’s and early 1970’s the disciplines of biochemistry and molecular biology 
 were wrought with questions pertaining to the double helical structure of DNA, as elucidated by J.D. Watson and F. Crick (1953). Of considerable interest at this time was the newfound idea of  whole chromosome-sized DNA molecules; that is, that one molecule of DNA might comprise anentire chromosome (Kavenoff 
et al 
, 1973). Evidence that DNA molecules were far longer thanpreviously believed led to the recognition of a DNA replication problem, first proposed by M.Delbrück (Delbrück, 1979; Holmes, 1998). Delbrück argued that strand separation of the DNAdouble helix during the process of replication would introduce considerable strain to the helix,inducing a supercoiled state. Indeed,
studies of polyoma viral DNA in the 1960’s led to the
discovery of the phenomenon of DNA supercoiling on the basis of observed anomalousfractionation patterns of covalently-closed circular DNA (Bates & Maxwell, 2005).DNA supercoiling is the result of topological strain on the DNA helix caused by over- orunder-winding of DNA. Two distinct [topo]isomeric forms of supercoiled DNA exist: positively and negatively supercoiled DNA. Positive supercoiling is the consequence of over-twisting the DNAhelix, while negative supercoiling results from helix unraveling. Central in considering thetopological characteristics of deformed DNA is the quantitative parameter
linking number 
(Lk), whichessentially describes the number of helical turns in a DNA molecule (i.e., the number of linkagesbetween the two strands). Because the helical structure of DNA is an intrinsic property of itschemical composition, a standard Lk° can be determined for a DNA molecule with a length
 N 
, suchthat Lk°=
 N 
/10.5, where 10.5 is the number of base pairs per helical turn of DNA under standardbiological conditions. Thereby, negatively supercoiled DNA is any in which Lk is less than Lk° andlikewise, Lk is greater than Lk° describes positively supercoiled DNA (Bates & Maxwell, 2005; Wang, 1998).It is evident that a host of topological considerations influence the myriad cellular processesinvolving DNA. Therefore, it comes as no surprise that DNA topology is highly regulated throughcatalytic protein interactions. Specifically, the interconversion of isomeric DNA topologies isfacilitated by the family of enzymes called DNA topoisomerases (topos). In a general sense,topoisomerases catalyze the interpenetration of DNA strands or individual DNA molecules,indirectly coupled to the energy of ATP hydrolysis or the energy of supercoiling itself (Wang, 1998). Topoisomerase was first purified from
 E. coli 
by J.C. Wang (1971) and coined
ω
. In his pioneering 
 
 work, Wang observed that incubation of 
ω
with negative superhelical DNA reduced the number of superhelical turns (Wang, 1971). Today 
ω
protein is known as topo IA (Corbett & Berger, 2004). Topoisomerase enzymes comprise a diverse protein superfamily with multiple constituentsfound in all organisms, from mammals to plants to archaea, as well as certain viruses. Two primary classes of topoisomerases are defined: type I and type II enzymes. These two categories of toposdiffer in three distinct respects: (a) type I enzymes are solely capable of single strand cleavage, whereas type II enzymes catalyze double strand cleavage (corresponding to a discrete change in Lk of ±2/catalytic cycle, for type II). (b) The catalytic mechanism of type I enzymes is independent of  ATP, while type II enzymes are ATP-dependent (Corbett & Berger, 2004). (c) Type I topos arecapable of knotting/unknotting circular ssDNA, catalyzing circular duplex formation, andcatenation/decatenation of nicked DNA circles, whereas type II enzymes are capable of knotting/unknotting circular dsDNA, and inducing catenation/decatenation between intact circularduplexes (Bates & Maxwell, 2005). Topoisomerases are further divided into subfamilies IA, IB, IIA, and IIB. Type IA enzymesinclude eubacterial topoI and topoIII, yeast topoIII, mammalian topoIII
α
and
β
, and reverse gyrase.Nearly all members of this subfamily exhibit monomeric subunit architecture (excluding 
 M. kandleri 
reverse gyrase). Furthermore, IA topos are mechanistically defined by the stabilization of strandcleavage through the attachment of a 5' phosphate (as opposed to a 3' phosphate) to an active sitetyrosine residue, dependence on Mg(II), and exclusive substrate specificity for negative supercoils.Comparatively, examples of type IB enzymes include eukaryotic topoI, Pox virus topo, andeubacterial topoV. The type IB subfamily differs from type IA in that no metal cofactor is required,both negative and positive supercoils can serve as substrates, and strand cleavage is stabilized by covalent attachment of a 3' phosphate to an active site tyrosine (Corbett & Berger, 2004).In contrast to type I topos, the type IIA subfamily comprise nearly all type II topos, with thesole exception of type IIB archaeal topoVI. IIA enzymes include eubacterial DNA gyrase (Gyr),eubacterial topo IV, yeast topoII, and mammalian topoII
α
and
β
. DNA Gyrase is of particularinterest due to its ability to induce negative supercoiling, in addition to relaxing supercoils. Yeast andmammalian type II topos are characterized by homodimeric subunit organization, while Gyr andtype IIB topoVI are heterotetramers of the structure A
2
B
2
and topoIV is a heterotetramer of theform C
2
E
2
(Champoux, 2001). The cellular roles of type II topos include the release of intertwinedchromosome pairs during mitosis, a consequence of their decatenation activity; interestingly,
 E. coli 
 cells lacking topoisomerase cannot survive (Wang, 1998). Furthermore, type II topoisomerases
 
possess intriguing structural and mechanistic features underlying their distinctive function; forexample, the role of ATP hydrolysis in enzyme catalysis is highly unique. Type IIA topoisomerases are comprised of three predominant functional regionsconsecutively joined together by flexible linkers: the amino-terminal ATPase dimeric region formedby the GHKL and transducer domains, the toprim and 5Y-CAP domains, and the C-terminalscaffolding/accessory domain (C-gate). The two ATPase N-terminal regions of the homodimerictopoII enzyme collectively form an ATP-operated clamp (N-gate) structure. The
enzyme’s
mid-region, composed of the conserved toprim and 5Y-CAP domains, plays a central role in DNAbinding and strand cleavage. The 5Y-CAP is so-
called because it contains a “winged
-
helix” DNA
binding fold found in
 E. coli 
Catabolite Activator Protein, as well as the catalytic Y(782) residue thatforms a phosphodiester linkage with the DNA 5' terminus following nucleophilic attack viatransesterification, hence 5Y-CAP. Additionally, an R residue adjacent to Y782 is thought tofunction in positioning the scissile bond. The toprim domain is so-named because this fold is foundin topoisomerases as well as primases. This domain is functionally characterized by an acidic clusterthought to act in chelating the Mg 
2+
cofactor (Corbett & Berger, 2004).Catalysis by type II topoisomerases occurs through a double-gate strand passage reactionmechanism, in which both strands of a dsDNA segment are cleaved and a second duplex segment ispassed through the break. This process may be either intramolecular, as in the case of superhelicalrelaxing, or intermolecular, as in the formation/deformation of catenanes. Firstly, the G (gate)segment is bound at the high affinity DNA binding folds of the CAP domains in a mannerdependent on an open N-gate conformation. G segment binding induces an increase in N-gatecycling between open and closed states, corresponding to ATP binding and ADP+P
i
release, untilthe T (transported) segment is captured (Wang, 1998). T-segment capture and N-gate closing arefollowed by transient cleavage of the G segment by the two homologous active site Y residues andopening of the DNA gate formed by the 5Y-CAP and scaffold domains. It has been suggested thatDNA gate opening is facilitated by steric repulsion of the T segment, whereby the T segment isdrawn toward the DNA gate by an electrostatic potential gradient. Indeed, quantitative analysis hasdemonstrated that the homodimer interface and the large cavity on the opposite side of the Gsegment are lined with positive charges, whereas the N-gate arms are highly acidic (Berger
et al 
 1996). Furthermore, it is thought that migration of the T segment into the central hole results in thecollapse of the DNA gate followed by processive constriction at the homodimer interface to support

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