Genetic and Biochemical Characterizations of aLhr1 Helicase in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

Abstract

Homologous recombination (HR) refers to the process of information exchange between homologous DNA duplexes and is composed of four main steps: end resection, strand invasion and formation of a Holliday junction (HJ), branch migration, and resolution of the HJ. Within each step of HR in Archaea, the helicase-promoting branch migration is not fully understood. Previous biochemical studies identified three candidates for archaeal helicase promoting branch migration in vitro: Hjm/Hel308, PINA, and archaeal long helicase related (aLhr) 2. However, there is no direct evidence of their involvement in HR in vivo. Here, we identified a novel helicase encoded by Saci_0814, isolated from the thermophilic crenarchaeon Sulfolobus acidocaldarius; the helicase dissociated a synthetic HJ. Notably, HR frequency in the Saci_0814-deleted strain was lower than that of the parent strain (5-fold decrease), indicating that Saci_0814 may be involved in HR in vivo. Saci_0814 is classified as an aLhr1 under superfamily 2 helicases; its homologs are conserved among Archaea. Purified protein produced in Escherichia coli showed branch migration activity in vitro. Based on both genetic and biochemical evidence, we suggest that aLhr1 is involved in HR and may function as a branch migration helicase in S. acidocaldarius.

The Impact of Single-Stranded DNA-Binding Protein SSB and Putative SSB-Interacting Proteins on Genome Integrity in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

“…Comprehensive phylogenetic analysis of the SF2 and aLhr helicases in living things indicated that SSO0017, SSO0394, and SSO0965 were divided into SftH, aLhr1, and aLhr3, respectively [39,40], but no research on SftH and aLhr3 homologs were reported for Archaea. We recently characterized SacaLhr1 helicase (Saci_0814) as an SSO0394 homolog in S. acidocaldarius [38], but it is not clear whether SacaLhr1 is functionally required for DNA repair. Regarding the three hypothetical proteins, McRobbie et al reported that the SSO2452 homolog was a recombination protein RecA paralog (Rad55) [41], and genetic analysis suggested that Rad55 (RadC1 encoded by SiRe_0240) was involved in DNA repair [42].…”

“…In our previous research, we constructed two deletion mutant strains of S. acidocaldarius for the gene encoding of SSB and SacaLhr1 from the parental strain DP-1 [37,38]. Using the gene knockout strategy from our past work [43], we constructed sftH-, alhr3-, Saci_0790-, and rad55-deletion strains by deleting each gene coding region from the parental strain DP-1 (Figure S1A).…”

“…To obtain direct evidence of the involvement of SSB in HR in vivo, we also examined HR frequency via the double-crossover of HR using a linear marker cassette (pyrElacS800) in ∆ssb. In this test, the selectable marker (lacS-pyrE) could only be maintained if it was integrated into the host genome by HR via double crossover between the linear marker cassette and the chromosome at the 5 and 3 homologous regions of the target locus (see Materials and Methods section) [38]. The autonomously replicating vector pSAV2 containing pyrE was used as a control to determine the transformation efficiency.…”

“…Further phenotypic characterization of the ssb-deleted strain was necessary to provide the genetic evidence described above. In addition, we previously identified a novel helicasearchaeal long helicase related (aLhr) 1, SacaLhr1, in S. acidocaldarius-that dissociated a synthetic Holliday junction (HJ) in vitro [38]. Notably, the HR frequency in the alhr1-deleted strain is five-fold lower than that in the parent strain, indicating that SacaLhr1 may be involved in HR in vivo [38].…”

“…In addition, we previously identified a novel helicasearchaeal long helicase related (aLhr) 1, SacaLhr1, in S. acidocaldarius-that dissociated a synthetic Holliday junction (HJ) in vitro [38]. Notably, the HR frequency in the alhr1-deleted strain is five-fold lower than that in the parent strain, indicating that SacaLhr1 may be involved in HR in vivo [38]. However, its physiological role in DNA repair needs to be further characterized.…”