HLTF’s Ancient HIRAN Domain Binds 3′ DNA Ends to Drive Replication Fork Reversal

Abstract

Summary Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3′-ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro, through its interaction with 3′-ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell.

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

“…6B, panel 12). Thus, HLTF deficiency per se is not sufficient to trigger DNA-damage checkpoint, as reported in previous studies (45,46), nor is HLTF required to mediate cell cycle arrest in G2 phase by Vpr.…”

“…Although integration competent HIV-1 preintegration complexes form in the cytoplasm, the completion of reverse transcription does not appear to be a prerequisite for HIV-1 entry to the nucleus (55), where partially reverse-transcribed viral cDNA can be accessed by DNA repair proteins. Alternatively, HLTF could modulate processing of the integrated proviral DNA, as it is known to control the choice between two pathways of postreplication repair of DNA lesions at the replication fork: error-free, by template switching, and error-prone, through recruitment of mutagenic translesion DNA polymerases (45,56). As these two distinct modes of DNA repair, error-free and error-prone, have different mutagenic outcomes upon repair of chromosomal DNA, they would also have different consequences for the fidelity of HIV-1 provirus repair, which provides another tentative rationale for the removal of HLTF by HIV-1 Vpr.…”

“…Similar experiments performed with U2OS HLTF-KO cells [U2OS.HLTF. KO-iH1.Vpr (45)] revealed that WT NL4-3 Vpr arrested these cells in G2 phase (Fig. 6B, panel 12).…”

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

“…One alternative and non-mutually exclusive possibility is that HLTF degradation is related to the ability of Vpr to interfere with the immune response and the cooperation between immune cells (77)(78)(79)(80)(81) is related to the ability of Vpr to escape or promote immune detection in primary cells should be investigated. A role of HLTF in DNA sensing could be a consequence of its capacity to bind ssDNA ends (82). It is also possible that HLTF is not a direct target of Vpr and that its degradation results from the inactivation of another cellular protein that was not detected in our SILAC experiments.…”

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

“…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).…”