The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression

Abstract

Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. The N-terminal HIRAN domain binds specifically to the 3'-end of single-stranded DNA (ssDNA), and disrupting this function interferes with fork regression as well as replication fork progression in cells under replication stress. Here, we investigated the mechanism by which the HIRAN-ssDNA interaction facilitates fork remodeling. Our results indicated that HIRAN capture of a denatured nascent leading 3'-end directs specific binding of HLTF to forks. DNase footprinting revealed that HLTF binds to the parental duplex ahead of the fork and at the leading edge behind the fork. Moreover, we found that the HIRAN domain is important for initiating regression of forks when both nascent strands are at the junction, but is dispensable when forks contain ssDNA regions on either template strand. We also found that HLTF catalyzes fork restoration from a partially regressed structure in a HIRAN-dependent manner. Thus, HIRAN serves as a substrate-recognition domain to properly orient the ATPase motor domain at stalled and regressed forks and initiates fork remodeling by guiding formation of a four-way junction. We discuss how these activities compare with those of two related fork remodelers, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) and zinc finger RANBP2 type-containing 3 (ZRANB3) to provide insight into their nonredundant roles in DNA damage tolerance.

Control of DNA Damage Bypass by Ubiquitylation of PCNA

“…The HIRAN domains of Rad5 and of HLTF are important for fork-remodeling activity. It has been proposed that the HIRAN domain is important for substrate recognition and positioning of the helicase domain on the DNA substrate [71].…”

“…The HIRAN domain of HLTF is required for unwinding forked DNA substrates containing double-stranded DNA on both the leading and the lagging strand of the fork without single-stranded gaps at the fork junction [71]. By contrast, this domain is not as important when unwinding forked DNA substrates with single-stranded gaps on either the leading or the lagging strand.…”

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

“…They are therefore believed to be important for inducing TS. In addition, HLTF mediates fork reversal via its HIRAN domain interacting with the 3'ends of a frayed fork [16]. For SHPRH, which lacks a HIRAN domain, fork reversal has not been reported yet.…”

“…The latter contains a functional PCNA interacting sequence, AlkB homolog 2 PCNA interacting motif (APIM) [17]. In addition, SMARCAL1, ZRANB3, and HLTF can restore a three-way-replication fork from a four-way-reversed replication fork [16,18]. Furthermore, both RAD5 homologs are reported to be involved in TLS: HLTF by recruiting POLη after UV-induced DNA damage, and SHPRH by recruiting POLκ after methyl methanesulfonate (MMS)-induced DNA damage [19].…”

The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on the DNA Lesion Type

“…HLTF and SHPRH are reported to suppress UV-or methyl methanesulfonate (MMS)-induced mutagenesis, respectively [7]. In addition, HLTF is an ATP-dependent translocase able to catalyze fork regression by its HIRAN domain [9]. Both proteins have been suggested as tumor suppressor genes, because dysregulation of HLTF and SHPRH is found in several types of cancers [10][11][12][13].…”