Human Topoisomerase IIIα Is a Single-stranded DNA Decatenase That Is Stimulated by BLM and RMI1

Abstract

Human topoisomerase III␣ is a type IA DNA topoisomerase that functions with BLM and RMI1 to resolve DNA replication and recombination intermediates. BLM, human topoisomerase III␣, and RMI1 catalyze the dissolution of double Holliday junctions into noncrossover products via a strand-passage mechanism. We generated single-stranded catenanes that resemble the proposed dissolution intermediate recognized by human topoisomerase III␣. We demonstrate that human topoisomerase III␣ is a single-stranded DNA decatenase that is specifically stimulated by the BLM-RMI1 pair. In addition, RMI1 interacts with human topoisomerase III␣, and the interaction is required for the stimulatory effect of RMI1 on decatenase activity. Our data provide direct evidence that human topoisomerase III␣ functions as a decatenase with the assistance of BLM and RMI1 to facilitate the processing of homologous recombination intermediates without crossing over as a mechanism to preserve genome integrity.Topoisomerases are ubiquitous enzymes conserved from bacteria to humans. Their roles in modulating DNA topology in replication, transcription, and other cellular processes (1, 2) make them indispensable for cell viability. There are four subfamilies of topoisomerases as follows: IA, IB, IIA, and IIB. Type IA topoisomerases change DNA topological states in discrete steps of one via an enzyme-bridging mechanism (1, 2). The catalytic tyrosine residue initiates a transesterification reaction in a single-stranded region to generate a transient DNA break, allowing for the passage of the intact strand through the break. After religation of the broken strand by a reversal of the reaction, the enzyme is free to engage in another round of catalysis (1, 2). Members of the type IA topoisomerase family include Escherichia coli topoisomerase I (EcTop1) and III (EcTop3), yeast topoisomerase III (Top3), and two isoforms of topoisomerase III, ␣ (Topo 2 III␣) and ␤ (Topo III␤), in higher eukaryotes. These enzymes exhibit high sequence similarity in the N-terminal catalytic core domain, whereas the C-terminal tails are variable (1). In addition to the ability to relax negatively supercoiled DNA, EcTop1 is capable of catalyzing knotting, unknotting, and interlinking of DNA substrates that contain exposed single-stranded regions (1). Because single-stranded DNA gaps are a common feature found at DNA replication forks, replication termination sites, and replication and repair sites, it is believed that the main function of type IA topoisomerases is to unlink DNA catenanes.Type IA topoisomerases function in concert with RecQ helicases to control recombination events (3). RecQ helicases are a highly conserved family of DNA helicases that are required for the maintenance of genome integrity (4). Human topoisomerase III␣ (hTopo III␣) physically interacts with BLM, one of the five RecQ helicases in humans (5, 6). Biallelic mutations of BLM give rise to a clinically defined cancer predisposition disorder, Bloom syndrome (BS) (7). BS cells display signs of genome instability, fea...

Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases

“…Top3α alone is capable of dissolving the network, and the presence of Blm/Rmi1/Rmi2 further stimulates this activity. Because only a single or a limited number of topological linkages connect two helices in a hemicatenane, it is reasonable to expect that the strand-passage activity of Top3α is responsible for the dissolution step, and the presence of Rmi1/Rmi2 or Rmi1/Rmi2 and Blm can stimulate this activity further in situations similar to previous observations regarding the relaxation activity of Top3α (34,35).This result adds additional evidence that the network is conjoined through hemicatenane linkages and that it can be dissolved by a type IA enzyme from either a prokaryotic or eukaryotic source.…”

RMI1 Promotes DNA Replication Fork Progression and Recovery from Replication Fork Stress

“…Biochemically, RMI1 stimulates TopoIII␣ activity in DHJ dissolution and in ssDNA decatenation (38,54,58). Stimulation requires a physical interaction between RMI1 and TopoIII␣ (39,50,58). At the cellular level, RMI1 is required for the recruitment of BLM to nuclear foci in response to DNA damage (59).…”

“…The primary role of RMI1 is thought to be the resolution of recombination intermediates in concert with BLM and TopoIII␣. Biochemically, RMI1 stimulates TopoIII␣ activity in DHJ dissolution and in ssDNA decatenation (38,54,58). Stimulation requires a physical interaction between RMI1 and TopoIII␣ (39,50,58).…”

“…The TopoIII␣-interacting domain of BLM is required for the suppression of SCEs in BS cells (26), suggesting that TopoIII␣ plays an antirecombinogenic role with BLM. Biochemically, TopoIII␣ catalyzes the decatenation of single-stranded DNA catenanes (58). The decatenase activity of TopoIII␣, coupled with the helicase activity of BLM, is uniquely suited to dissolve double-Holliday-junction (DHJ) structures, which arise during homologous recombination, via a strand passage mechanism to prevent the exchange between flanking sequences (55).…”

MTE1 Functions with MPH1 in Double-Strand Break Repair

“…Double Holliday junction (dHJ) intermediates that result from invasion of a homologous chromosome by both ends of a resected DSB (Szostak et al 1983) can be resolved nucleolytically by the action of the Yen1 and Mus81/Mms4 endonucleases (Blanco et al 2010;Ho et al 2010) to produce a random distribution of crossover and noncrossover products. By contrast, the same dHJ intermediates can be dissolved by the combined helicase and ssDNA decatenase action of the Bloom/TopIIIa/Rmi1 complex (Sgs1/Top3/Rmi1 in yeast) (Wu et al 2006;Yang et al 2010) to yield exclusively noncrossover products (Wu and Hickson 2003). Crossovers can also be prevented if the D-loop structure that results from the first strand invasion by one end of a resected DSB into the homologous chromosome is unwound before capture of the second end to form the dHJ.…”