Does PCNA diffusion on DNA follow a rotation-coupled translation mechanism?
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ABSTRACT:
SUBMITTER: Greenblatt HM
PROVIDER: S-EPMC7536400 | biostudies-literature | 2020 Oct
REPOSITORIES: biostudies-literature
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Does PCNA diffusion on DNA follow a rotation-coupled translation mechanism?
Greenblatt Harry Mark HM Rozenberg Haim H Daitchman Dina D Levy Yaakov Y
Nature communications 20201005 1
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Project description: Not available
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Project description:The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA.
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Project description:The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
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Project description:The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.
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Project description:DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA "braiding" experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements.
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Project description:BackgroundScapulothoracic upward rotation (UR) is an important shoulder complex motion allowing for a larger functional work space and improved glenohumeral muscle function. However, the kinematic mechanisms producing scapulothoracic UR remain unclear, limiting the understanding of normal and abnormal shoulder movements.ObjectiveThe objective of this study was to identify the coupling relationships through which sternoclavicular and acromioclavicular joint motions contribute to scapulothoracic UR.DesignThis was a cross-sectional observational study.MethodsSixty participants were enrolled in this study; 30 had current shoulder pain, and 30 had no history of shoulder symptoms. Shoulder complex kinematics were quantified using single-plane fluoroscopy and 2D/3D shape matching and were described as finite helical displacements for 30-degree phases of humerothoracic elevation (30 degrees-60 degrees, 60 degrees-90 degrees, and 90 degrees-120 degrees). A coupling function was derived to estimate scapulothoracic UR from its component motions of acromioclavicular UR, sternoclavicular posterior rotation, and sternoclavicular elevation as a function of acromioclavicular internal rotation. The proportional contributions of each of the component motions were also calculated and compared between phases of humerothoracic elevation and groups.ResultsScapulothoracic UR displacement could be effectively predicted using the derived coupling function. During the 30- to 60-degree humerothoracic elevation phase, acromioclavicular UR accounted for 84.2% of scapulothoracic UR, whereas sternoclavicular posterior rotation and elevation each accounted for < 10%. During later phases, acromioclavicular UR and sternoclavicular posterior rotation each accounted for 32% to 42%, whereas sternoclavicular elevation accounted for < 11%.LimitationsError due to the tracking of sternoclavicular posterior rotation may have resulted in an underprediction of its proportional contribution and an overprediction of the proportional contribution of acromioclavicular UR.ConclusionsAcromioclavicular UR and sternoclavicular posterior rotation are the predominant component motions of scapulothoracic UR. More research is needed to investigate how these coupling relationships are affected by muscle function and influenced by scapular dyskinesis.
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