Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

Abstract

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3 0 end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3 0 -terminal nucleotide residue of DNA. The interaction with the deoxyribose 3 0 -OH is essential for the 3 0 -terminal recognition. In contrast, the direct interaction with 3 0 -end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3 0 end. Thus, the N-terminal domain of PriA recognizes the 3 0 -end base in a base-non-selective manner, in addition to the deoxyribose and 5 0 -side phosphodiester group, of the 3 0 -terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

Structural mechanisms of PriA-mediated DNA replication restart

“…The first is the 3′BD, which has been shown previously to bind the 3′ end of the leading-strand arm of replication fork structures (26). This recognition is thought to help direct PriA replisomal reloading activity to appropriate DNA structures (25)(26)(27)(28). The 3′BD structure from full-length KpPriA is strikingly similar to the previously determined structure of the isolated 3′BD from EcPriA (26) (rmsd = 1.8 Å for 90 common C α atoms).…”

“…The arrangement of DBDs within the PriA structure complements the positions of the three DNA arms of branched replication fork and D-loop structures, which could facilitate structure-specific DNA binding by PriA. Within this array of domains, we propose that the 3′BD would recognize the 3′ end of the leading strand as has been observed previously (26), whereas the HD would preferentially bind to lagging-strand DNA. Lagging-strand binding by the HD in this model is consistent with PriA's noted 3′-5′ translocase and helicase activities and its preference for unwinding the nascent lagging strand of replication fork structures (11-13, 35, 36).…”

“…The structure of KpPriA showed that the DBD is subdivided into two DNA-binding elements. The first is the 3′BD, which has been shown previously to bind the 3′ end of the leading-strand arm of replication fork structures (26). This recognition is thought to help direct PriA replisomal reloading activity to appropriate DNA structures (25)(26)(27)(28).…”

Replication Restart in Bacteria

“…A full-length structure of PriA from K. pneumoniae with 88% identity with E. coli PriA and the ability to complement PriA mutants of E. coli revealed a structure with six tightly clustered domains (53). The first domain has been crystallized before from the E. coli protein and shown to be able to bind the 3=-OH group of ssDNA (41). The next is a winged helix domain that is capable of binding doublestranded DNA (dsDNA).…”

“…Surprisingly, the recognition of different structures by these two proteins in vitro is at odds with a redundant function in vivo, unless each of these structures can be derived from the other one. PriA recognition of DNA 3= ends at forks is mediated by its N-terminal domain (41). PriA binds PriB in the helicase domain, while PriB has overlapping binding sites for DnaT and ssDNA (42).…”