Coordinated protein and DNA remodeling by human HLTF on stalled replication fork

Abstract

Human helicase-like transcription factor (HLTF) exhibits ubiquitin ligase activity for proliferating cell nuclear antigen (PCNA) polyubiquitylation as well as double-stranded DNA translocase activity for remodeling stalled replication fork by fork reversal, which can support damage bypass by template switching. However, a stalled replication fork is surrounded by various DNA-binding proteins which can inhibit the access of damage bypass players, and it is unknown how these proteins become displaced. Here we reveal that HLTF has an ATP hydrolysis-dependent protein remodeling activity, by which it can remove proteins bound to the replication fork. Moreover, we demonstrate that HLTF can displace a broad spectrum of proteins such as replication protein A (RPA), PCNA, and replication factor C (RFC), thereby providing the first example for a protein clearing activity at the stalled replication fork. Our findings clarify how remodeling of a stalled replication fork can occur if it is engaged in interactions with masses of proteins. U nrepaired DNA lesions are dangerous obstacles for the replication machinery because most of them cannot be accommodated into the active site of the replicative polymerases, thereby blocking the progression of the replication fork. Prolonged stalling might lead to DNA strand breaks, chromosomal rearrangements, or cell death (1-3). To minimize this danger cells have evolved various DNA damage bypass mechanisms that are initiated by exchanging protein components of the normal replication machinery for protein players which either carry out a direct damage bypass or manipulate the stalled fork to generate transitional DNA structures that facilitate damage bypass indirectly. In the first situation, specialized translesion synthesis polymerases that can accommodate even bulky lesions at their active sites take over the 3′ primer end from the accurate replicative polymerase and incorporate either a correct or an incorrect nucleotide opposite the lesion (4, 5). Alternatively, DNA remodeling might lead to the annealing of the stalled nascent strand to the newly synthesized strand of the undamaged sister duplex, resulting in template switch (6-9). Although the exact mechanism and factors of template switch have remained largely unknown, two proposed mechanisms have gained significant attention. One is named-based on the shape of the intermediate DNA structure-chicken foot model, which proposes pairing of the two newly synthesized strands of the sister chromatids by reversal of the stalled fork (9, 10). The other model also suggests pairing of the newly synthesized strands, but assumes that it occurs via a D-loop recombination intermediate (6,(11)(12)(13). It is possible that these mechanisms are not mutually exclusive and the choice can be regulated at the level of displacement and exchange of the protein components of the stalled replication machinery for various new players.It is generally accepted that in eukaryotic cells damage bypass is governed by Rad6 and Rad18, a ubiquitin conjugating and a ...

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“…Both enzymes have been reported to be able to create and resolve HR intermediates such as D-loops independent of RAD51, which may provide primers for the repair of gaps generated during replication of damaged DNA (62,63). A major function of HLTF appears to be the promotion of fork reversal upon replication block (43,64,65). ZRANB3, a SWI/ SNF catalytic subunit (SNF2) DNA translocase like HLTF, has been proposed to cooperate with HLTF in the remodeling of the blocked fork, additionally contributing a structure-specific endonuclease for the fork remodeling (45,66,67).…”

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

“…Rad54) dislodge proteins bound to double-stranded DNA leading to the disassembly of toxic recombination intermediates or static dead-end complexes (58,59). The human helicase-like transcription factor coordinates remodeling of stalled replication forks by displacing the clamp proliferating cell nuclear antigen or clamp loader RFC (60). Although these studies suggest a potential role of helicase-like proteins to dislodge proteins bound to DNA, very little is known about the mechanism, regulation, and biological significance of helicase-catalyzed protein displacement, especially in eukaryotes.…”