Biochemical activities and genetic functions of the Drosophila melanogaster Fancm DNA helicase in DNA repair

Repair of DNA damage is essential to the preservation of genomic stability. During repair of double-strand breaks, several helicases function to promote accurate repair and prevent the formation of crossovers through homologous recombination. Among these helicases is the Fanconi anemia group M (FANCM) protein. FANCM is important in the response to various types of DNA damage and has been suggested to prevent mitotic crossovers during double-strand break repair. The helicase activity of FANCM is believed to be important in these functions, but no helicase activity has been detected in vitro. We report here a genetic and biochemical study of Drosophila melanogaster Fancm. We show that purified Fancm is a 3ʹ to 5ʹ ATP-dependent helicase that can disassemble recombination intermediates, but only through limited lengths of duplex DNA. Using transgenic flies expressing full-length or truncated Fancm, each with either a wild-type or mutated helicase domain, we found that there are helicase-independent and C-terminus-independent functions in responding to DNA damage and in preventing mitotic crossovers. INTRODUCTION DNA helicases are a diverse group of enzymes that separate the two strands of duplex DNA. Using the free energy derived from the hydrolysis of a 5ʹ -nucleoside triphosphate, generally ATP, the helicase catalyzes the unwinding of duplex DNA to yield single stranded DNA (ssDNA), a process that is required in replication, transcription, recombination, and repair. Thus, helicases are involved in essentially all metabolic pathways that require the separation of duplex DNA (Brosh 2013; Khan et al. 2015). Helicases exhibit a diversity of structure and mechanism that may be related to the often unique and specialized roles that these enzymes can play in the cell (Brosh 2013; Daley et al. 2013). Importantly, distinct helicases can interact with specific DNA substrates. For example, during repair of DNA damage different helicases often act within particular pathways and on unique DNA intermediates that are generated as repair progresses, such as Holliday junctions (HJs) or displacement loops (D-loops). This can be observed in the requirement for helicases to recognize and act on specific DNA structures during the process of double-strand break (DSB) repair via homologous recombination (HR). DSB repair by HR is a complex process with several key events: resection of the 5ʹ end at the strand break; invasion of the Rad51-coated 3ʹ ssDNA tail into a homologous duplex sequence, generating a displacement loop (D-loop); DNA synthesis primed from the invading 3ʹ end; and resolution into one of either two types of recombination product crossovers (COs) or non-crossovers (NCOs). The formation of COs during DSB repair in mitotically dividing cells can be hazardous as they can result in loss of heterozygosity and gross chromosomal rearrangements (Lorenz and Whitby 2006; Andersen and Sekelsky 2010). Therefore, prevention Genetics: Early Online, published on July 29, 2016 as 10.1534/genetics.116.192534