The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA

Abstract

HepA-related protein (HARP) (also known as SMAR-CAL1) is an ATP-driven annealing helicase that catalyzes the formation of dsDNA from complementary Replication protein A (RPA)-bound ssDNA. Here we find that HARP contains a conserved N-terminal motif that is necessary and sufficient for binding to RPA. This RPAbinding motif is not required for annealing helicase activity, but is essential for the recruitment of HARP to sites of laser-induced DNA damage. These findings suggest that the interaction of HARP with RPA increases the concentration of annealing helicase activity in the vicinity of ssDNA regions to facilitate processes such as DNA repair.Supplemental material is available at http://www.genesdev.org.Received June 12, 2009; revised version accepted August 7, 2009. Proteins in the SNF2 family of ATPases participate in a variety of nuclear processes, such as chromatin assembly and remodeling, transcription, DNA repair, and recombination (Gorbalenya and Koonin 1993;Eisen et al. 1995;Flaus et al. 2006). The HepA-related protein (HARP, also known as SMARCAL1) is a distant member of the SNF2 family (Coleman et al. 2000;Flaus et al. 2006). HARP is present in many eukaryotes but appears to be absent in fungi. In humans, mutations in HARP contribute to the pleiotropic disorder known as Schimke immunoosseous dysplasia (SIOD) (Boerkoel et al. 2002).HARP is an ATP-dependent annealing helicase (Yusufzai and Kadonaga 2008). Specifically, HARP is able to rewind complementary ssDNA that is bound by the ssDNA-binding protein Replication protein A (RPA). HARP binds preferentially to fork DNA relative to ssDNA or to dsDNA (Yusufzai and Kadonaga 2008). The ATPase activity of HARP is also stimulated preferentially by fork DNA relative to ssDNA or dsDNA (Yusufzai and Kadonaga 2008). This property is consistent with the observation that the ATPase activity of HARP is enhanced by DNA species with ssDNA-dsDNA junctions (Hockensmith et al. 1986;Muthuswami et al. 2000). These findings suggest a model wherein the binding of HARP to a DNA fork activates its ATP-driven motor with which it catalyzes the rewinding of DNA.The DNA rewinding activity of HARP could potentially participate in many different processes such as transcription, DNA replication, and DNA repair, in which ssDNA regions are generated by the action of helicases or polymerases (for example, see Liu and Wang 1987;Kowalski et al. 1988;Havas et al. 2000;Pyle 2008). These ssDNA regions can be stabilized by ssDNAbinding proteins (SSBs) such as RPA, the major SSB in eukaryotes (for example, see Wold 1997;Iftode et al. 1999;Zou et al. 2006). RPA is a heterotrimer (RPA1 [70 kDa], RPA2 [32 kDa], and RPA3 [14 kDa]) that binds stably to ssDNA and prevents complementary DNA from reannealing. RPA is required for many cellular processes, including replication, recombination, and repair, during which it stabilizes ssDNA intermediates. Because HARP catalyzes the regeneration of dsDNA from complementary RPA-bound ssDNA, it is possible that there is a specific link between HARP and RPA...

hPrimpol1/CCDC111 is a human DNA primase‐polymerase required for the maintenance of genome integrity

“…We propose that, in response to replication stress, replication forks would be stalled and allow the formation of ssDNA regions that become coated with RPA. Through the interaction with RPA1, hPrimpol1, SMARCAL1/HARP [5][6][7][8][9] and/or other RPAbinding proteins are recruited to these stalled replication forks and exerts their enzymatic activities to reinitiate DNA replication and thus to limit replication-associated DNA damage (Fig 5G). …”

Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

“…This interaction between HARP and RPA is not required for annealing helicase activity, but rather serves to recruit HARP to sites of RPA-bound ssDNA that are formed during DNA damage or replicative stress (5)(6)(7)(8). Cells with reduced levels of HARP also exhibit increased levels of RPA-bound ssDNA during S phase relative to that seen with wild-type cells (5,7).…”

“…In addition to its ability to function as an annealing helicase, HARP binds directly to RPA via a conserved N-terminal motif (5)(6)(7)(8)(9). This interaction between HARP and RPA is not required for annealing helicase activity, but rather serves to recruit HARP to sites of RPA-bound ssDNA that are formed during DNA damage or replicative stress (5)(6)(7)(8).…”

Transcriptional and posttranscriptional mechanisms contribute to the dysregulation of elastogenesis in Schimke immuno-osseous dysplasia

“…SMARCAL1 could alter the local structure of DNA thereby promoting or inhibiting the transcription of specific genes via a direct effect of SMARCAL1 deficiency on the gene of interest (e.g., ELN) or from an indirect effect of SMARCAL1 deficiency on upstream pathways (e.g., increased expression of miR-29 family members, altered expression of the pathway regulating ELN poly(A) tail length, etc.). We hypothesize that SMARCAL1 deficiency mediates these effects by altering the epigenetic landscape as a consequence of its role in the DNA damage response (4,5) and replication fork restart (6)(7)(8). Replication fork stalling is associated with epigenetic instability (36); consequently, if SMARCAL1 replication fork stalling occurred near ELN or key regulators of ELN and ELN mRNA stability, the resulting epigenetic changes might produce the pathogenic gene expression alterations observed in the SIOD aorta.…”

“…SMARCAL1 functions as a DNA-dependent annealing helicase (2) that recognizes single-stranded to double-stranded DNA transitions and binds to transcriptionally active chromatin (3). SMARCAL1 is involved in the DNA stress response (4,5), the reactivation of stalled replication forks (6)(7)(8), and the modulation of gene expression (3). Key manifestations of the disease include growth failure due to spondyloepiphyseal dysplasia, renal failure due to focal segmental glomerulosclerosis, T-cell immunodeficiency, facial dysmorphism, and hyperpigmented macules (9).…”