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

Abstract

The structure and integrity of DNA is of considerable biological and biomedical importance, and it is therefore critical to identify and to characterize enzymes that alter DNA structure. DNA helicases are ATP-driven motor proteins that unwind DNA. Conversely, HepA-related protein (HARP) protein (also known as SMARCAL1 and DNA-dependent ATPase A) is an annealing helicase that rewinds DNA in an ATP-dependent manner. To date, HARP is the only known annealing helicase. Here we report the identification of a second annealing helicase, which we term AH2, for annealing helicase 2. Like HARP, AH2 catalyzes the ATP-dependent rewinding of replication protein A (RPA)-bound complementary singlestranded DNA, but does not exhibit any detectable helicase activity. Unlike HARP, however, AH2 lacks a conserved RPA-binding domain and does not interact with RPA. In addition, AH2 contains an HNH motif, which is commonly found in bacteria and fungi and is often associated with nuclease activity. AH2 appears to be the only vertebrate protein with an HNH motif. Contrary to expectations, purified AH2 does not exhibit nuclease activity, but it remains possible that AH2 contains a latent nuclease that is activated under specific conditions. These structural and functional differences between AH2 and HARP suggest that different annealing helicases have distinct functions in the cell. T he unwinding of dsDNA occurs frequently during normal cellular processes such as replication, repair, and transcription (for reviews, see refs. 1 and 2). In addition, ssDNA bubbles can arise spontaneously. Because unwound DNA can be stabilized by ssDNA-binding proteins such as replication protein A (RPA), there is considerable potential for the formation of stable RPA-bound ssDNA bubbles.HepA-related protein (HARP, also known as SMARCAL1) is an ATP-driven motor protein that rewinds RPA-bound complementary ssDNA strands (3). Because HARP anneals DNA, it is termed an annealing helicase. HARP binds preferentially to fork DNA relative to ssDNA or dsDNA, and the binding of HARP to fork DNA activates its ATPase activity. In humans, mutations in the HARP gene are responsible for a rare autosomal recessive pleiotropic disorder known as Schimke immunoosseous dysplasia, which typically leads to death in early childhood (4). It thus appears that the annealing helicase activity of HARP is essential for normal growth and development.In addition to its ability to function as an annealing helicase, HARP binds directly to RPA via a conserved N-terminal motif (5-9). 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 (5-8). 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). These findings suggest an important role for HARP in DNA structure and dynamics.To date, HARP is the only known annealing helicase. In these studies, we foun...

ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response

“…3B;Supplemental Fig. 3E,F;Yusufzai and Kadonaga 2010). Importantly, the ATPase activity was abolished by the K65R mutation, whereas the H1021A mutant retained activity comparable with wild-type ZRANB3.…”

“…2E). Given that the nuclease activity of ZRANB3 protein could not be detected in Escherichia coli genomic DNA (Yusufzai and Kadonaga 2010), it has been hypothesized that either the HNH domain has a non-nuclease-related function or its activity is activated in specific conditions. Our data reveal that the latter is the case and, surprisingly, that the activation of the endonuclease function necessitates ATPase activity contained within the helicase core of the ZRANB3 protein.…”

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication

“…Paradoxically, the closest homolog of SMARCAL1 in humans, annealing helicase 2 (AH2, also known as ZRANB3), also has annealing helicase activity despite a different domain structure and no unambiguous HARP domains (Yusufzai and Kadonaga 2010).…”

Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3)

“…SMARCAL1 and ZRANB3 are both annealing helicases that re-anneal complementary DNA strands (25,31) and catalyze replication fork remodeling reactions (4,5). The SMARCAL1 HARP2 domain is required for SMARCAL1 to bind DNA, hydrolyze ATP, anneal DNA, and remodel replication forks (4,20).…”