Biochemical Activities and Genetic Functions of the Drosophila melanogaster Fancm Helicase in DNA Repair

Abstract

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-terminal-independent functions in responding to DNA damage and in preventing mitotic crossovers. KEYWORDS biochemistry; genetics; DNA helicase; homologous recombination; synthesis-dependent strand annealing; ATP activity; crossing over D NA 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 D-loop; DNA synthesis primed from the invading 3ʹ end; and resolution into one of either two types of recombination product-crossovers (COs) or noncrossovers (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 o...

Annealing of Complementary DNA Sequences During Double-Strand Break Repair inDrosophilaIs Mediated by the Ortholog of SMARCAL1

“…Marcal1 K275M mutants had 67% white-eyed progeny, which was significantly reduced compared to heterozygotes but not significantly different from Marcal1 null mutants ( Figure 5F). * P = 0.0185; **** P , 0.0001. a key mediator of D-loop disassembly (Adams et al 2003;McVey et al 2004b) and recent studies suggest Fancm may play a minor role in this step as well (Kuo et al 2014;Romero et al 2016), though studies in human cells do not show a role for FANCM in SDSA (Zapotoczny and Sekelsky 2017). We did not observe phenotypes suggesting defects in D-loop dissociation in Marcal1 mutants, suggesting that the role of Marcal1 is downstream of D-loop dissociation.…”

FANCM regulates repair pathway choice at stalled replication forks

“…At the cellular level, FANCM homologs contribute to the non-crossover “synthesis-dependent strand annealing” (SDSA) pathway of HR (Paques and Haber, 1999; Prakash et al, 2009; Sun et al, 2008). FANCM homologs suppress meiotic and mitotic crossing over (Crismani et al, 2012; Knoll et al, 2012; Romero et al, 2016) including sister chromatid exchange (SCE), a cytological measure of crossing over (Bakker et al, 2009; Deans and West, 2009; Rosado et al, 2009). FANCM ATP hydrolysis mutants are defective for ICL resistance, indicating that FANCM motor function contributes to stalled fork repair (Nandi and Whitby, 2012; Xue et al, 2008).…”

The Drosophila Mutagen-Sensitivity Gene mus109 Encodes DmDNA2

“…For each vial, relative survival was calculated as the ratio of mus109 mutant to non-mutant flies in Brood 2, normalized to the same ratio in the corresponding Brood 1 vial. Vials with fewer than 15 progeny in either Brood 1 or 2 were excluded from analysis, as in [ 29 ]. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons.…”