Abstract
RecG catalyzes reversal of stalled replication forks in response to replication stress in bacteria. The protein contains a fork recognition (“wedge”) domain that binds branched DNA and a superfamily II (SF2) ATPase motor that drives translocation on double-stranded (ds)DNA. The mechanism by which the wedge and motor domains collaborate to catalyze fork reversal in RecG and analogous eukaryotic fork remodelers is unknown. Here, we used electron paramagnetic resonance (EPR) spectroscopy to probe conformational changes between the wedge and ATPase domains in response to fork DNA binding by Thermotoga maritima RecG. Upon binding DNA, the ATPase-C lobe moves away from both the wedge and ATPase-N domains. This conformational change is consistent with a model of RecG fully engaged with a DNA fork substrate constructed from a crystal structure of RecG bound to a DNA junction together with recent cryo-electron microscopy (EM) structures of chromatin remodelers in complex with dsDNA. We show by mutational analysis that a conserved loop within the translocation in RecG (TRG) motif that was unstructured in the RecG crystal structure is essential for fork reversal and DNA-dependent conformational changes. Together, this work helps provide a more coherent model of fork binding and remodeling by RecG and related eukaryotic enzymes.
Highlights
Faithful DNA replication at every round of cell division is critical for transmission of genetic information
Using a combination of electron paramagnetic resonance (EPR) spectroscopy and mutagenesis, we found that T. maritima RecG undergoes a conformational change in the ATPase motor relative to the wedge domain upon binding a model DNA replication fork
Recent cryo-electron microscopy (EM) structures of chromatin remodeling complexes CHD1, SNF2, INO80 bound to nucleosomes [40,41,42,43,44] and of Xeroderma pigmentosum B (XPB) helicase within the TFIIH component of the transcription pre-initiation complex [43] showed a conserved path of DNA across the N- and C-terminal lobes of the ATPase in a manner predicted from an archaeal Rad54 homolog bound to DNA in an open conformation [45]
Summary
Faithful DNA replication at every round of cell division is critical for transmission of genetic information. Stalled replication forks can lead to replisome disassembly, strand breaks and other pathogenic DNA structures, and are a potential source of genome instability associated with a number of diseases [1,2]. One important mechanism for stabilizing or restarting stalled forks is fork reversal (or fork regression), in which specialized motor proteins push the fork backward to convert the three-way fork into a four-way junction (Figure 1a) [5,6,7,8]. The Holliday junction-like structure serves as an important intermediate for recombination-coupled repair and can promote template switching to enable DNA synthesis from an unhindered nascent strand template [3]. Fork reversal may promote excision repair of fork-stalling DNA lesions by sequestering them away from the fork and back into the context of dsDNA
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