Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

Ineke Brouwer, Hongshan Zhang, Andrea Candelli, Davide Normanno, Erwin J.G. Peterman, Gijs J.L. Wuite, Mauro Modesti

Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

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Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin

Arjun S. Adhikari, Kristina B. Kooiker, Saswata S. Sarkar, Chao Liu, Daniel Bernstein, James A. Spudich, Kathleen M. Ruppel

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

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α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle

Lu Ma, Yuhao Kang, Junyi Jiao, Aleksander A. Rebane, Hyo Keun Cha, Zhiqun Xi, Hong Qu, Yongli Zhang

Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

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Kinesin’s Front Head Is Gated by the Backward Orientation of Its Neck Linker

Merve Yusra Dogan, Sinan Can, Frank B. Cleary, Vedud Purde, Ahmet Yildiz

Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos toward the plus-end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves neck linker (NL), a short peptide that interconnects the heads, but it remains unclear whether gating is facilitated by the NL orientation or tension. Using optical trapping, we measured the force-dependent microtubule release rate of kinesin monomers under different nucleotide conditions and pulling geometries. We find that pulling NL in the backward direction inhibits nucleotide binding and subsequent release from the microtubule. This inhibition is independent of the magnitude of tension (2–8 pN) exerted on NL. Our results provide evidence that the front head of a kinesin dimer is gated by the backward orientation of its NL until the rear head releases from the microtubule.

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Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

Ohad Iosefson, Adrian O. Olivares, Tania A. Baker, Robert T. Sauer

In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.

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In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

Géraldine Farge, Majda Mehmedovic, Marian Baclayon, Siet M.J.L. van den Wildenberg, Wouter H. Roos, Claes M. Gustafsson, Gijs J.L. Wuite, Maria Falkenberg

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.

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