Organization of Chromosomal DNA by SMC Complexes

Structural maintenance of chromosomes (SMC) complexes are key organizers of chromosome architecture in all kingdoms of life. Despite seemingly divergent functions, such as chromosome segregation, chromosome maintenance, sister chromatid cohesion, and mitotic chromosome compaction, it appears that these complexes function via highly conserved mechanisms and that they represent a novel class of DNA translocases.

S. pombe DNA translocases Rrp1 and Rrp2 have distinct roles at centromeres and telomeres that ensure genome stability

ABSTRACTHomologous recombination (HR) is a DNA repair mechanism that ensures, together with heterochromatin machinery, the proper replication, structure and function of telomeres and centromeres that is essential for the maintenance of genome integrity. Schizosaccharomyces pombe Rrp1 and Rrp2 participate in HR and are orthologues of Saccharomyces cerevisiae Uls1, a SWI2/SNF2 DNA translocase and SUMO-Targeted Ubiquitin Ligase. We show that Rrp1 or Rrp2 upregulation leads to chromosome instability and growth defects. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2. Either Rrp1 or Rrp2 overproduction results in a reduction in global histone levels, suggesting that Rrp1 and Rrp2 may modulate nucleosome dynamics. In addition we show that Rrp2, but not Rrp1, acts at telomeres. We propose that this role depends on the previously described interaction between Rrp2 and Top2. We conclude that Rrp1 and Rrp2 have important roles for centromere and telomere function and maintenance, contributing to the preservation of genome stability during vegetative cell growth.SUMMARY STATEMENTSchizosaccharomyces pombe DNA translocases Rrp1 and Rrp2 modulate centromere and telomere maintenance pathways and dysregulation of their activity leads to genome instability.

Advances in understanding DNA processing and protection at stalled replication forks

The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.

Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression

The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.

Simulations and Electrostatic Analysis Suggest an Active Role for DNA Conformational Changes During Genome Packaging by Bacteriophages

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens, as well as dsDNA bacteriophages – viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is, instead, an active component of the machinery. Computational studies on dsDNA in the channel of viral portal proteins reported here reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer (“stretched”) in regions of high negative electrostatic potential, and shorter (“scrunched”) in regions of high positive potential. These results suggest a mechanism that couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal and dsDNA work synergistically to promote genome packaging.

Single-Molecule Tracking of DNA Translocases inBacillus subtilisReveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA

ABSTRACTLike many bacteria,Bacillus subtilispossesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: anftsAdeletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzymein vivo. Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCEDNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane inB. subtilisand does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailedin vivostudy of DNA translocases at the single-molecule level.

Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA-Packaging Motors with Revolving Mechanism

The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Recently discovered was a third type of biomotor, including the viral DNA-packaging motor, beside the bacterial DNA translocases, that uses a revolving mechanism without rotation. By analogy, rotation resembles the Earth's rotation on its own axis, while revolving resembles the Earth's revolving around the Sun (see animations athttp://rnanano.osu.edu/movie.html). Herein, we review the structures of viral dsDNA-packaging motors, the stoichiometries of motor components, and the motion mechanisms of the motors. All viral dsDNA-packaging motors, including those of dsDNA/dsRNA bacteriophages, adenoviruses, poxviruses, herpesviruses, mimiviruses, megaviruses, pandoraviruses, and pithoviruses, contain a high-stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometries of target complexes that work sequentially.