Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex

Abstract

We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori c ), leaving stem-loop I and the adjacent 5 CTG 3, the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson Primase is required to initiate DNA replication in prokaryotes and eucaryotes. It functions to synthesize short primer RNAs (pRNA) on the separated strands of the replication fork, which provide primed templates with free 3Ј-OH terminus for DNA polymerase to elongate and synthesize the complementary DNA strand (4,13,14,25). Several prokaryotic priming systems have been established to study Escherichia coli primase (referred to as primase in this paper) function in vitro, such as cloned E. coli chromosomal origin of DNA replication (oriC) (9, 24), the origin of complementary DNA strand synthesis (ori c ) of single-stranded (ss) E. coli phages (e.g., X174[1]; G4 [3,25]; and st-1, ␣3, and K [2, 17]), double-stranded phage (e.g., [26]), and E. coli plasmid origins of DNA replication (e.g., R1 [11]). The simplest system is ss phage ori c exemplified by phage G4. In this system, primase in association with E. coli ss DNA-binding protein (SSB) synthesizes 29 nucleotides (nt) of pRNA from a unique 5Ј CTG 3Ј sequence in a region of the viral DNA strand called G4ori c (10). This is in contrast to other priming systems in which primase acts in concert with several other initiation proteins and synthesizes pRNA at many sites on the template DNA, although almost always initiating at 5Ј CTG 3Ј sequence (27). The G4ori c has been extensively studied as a model origin of DNA replication. Deletion studies have shown that the G4ori c region is a 140-nt, noncoding sequence containing potential secondary structure: stem-loops I, II, and III (5). Primase synthesizes pRNA at a 5Ј CTG 3Ј site, close to the 3Ј side of stem-loop I (7,8,18). Initiation of DNA replication in G4-related ss phages ␣3, st-1, and K (17) is similar to that of G4ori c .Several early studies provided information on the interaction of primase with the ori c DNA and SSB. A nuclease footprinting study on the interaction of primase with SSB-coated Kori c demonstrated that regions of the stem-loop structure were involved in primase binding (16). Then a stoichiometric study using gel filtration reported that two primase molecules bound on one G4ori c that was cloned into phage M13 ss-DNA (19). In a more recent study (21) to dissect the interactions of SSB and primase with G4ori c , we have demonstrated that two SSB tetramers bound on G4ori c in a fixed position. This resulted in the formation of a unique structure with two SSB tetramers (i.e., an SSB octamer) bound to the stem-loop region and other SSB tetramers bound on the 5Ј and 3Ј sides, leaving approximately 30 nt of free uncoated ss-DNA between the central SSB octamer and the adjacent SSB tetramers (or octamers). The 5Ј CTG 3Ј pRNA initiation site was located in the SSB-free linke...

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

“…Far Western analysis performed with HiSSBΔC failed to show any positive interaction with RecJ, as well as with RNA polymerase (data not shown), suggesting that the interaction between RecJ and SSB was mediated through the C-terminus of SSB. The C-terminus of SSB proteins is known to mediate the direct interaction of SSB with a number of DNA-transacting proteins, including E. coli ExoI, 35 RecA, 22 RNA polymerase, 41 primase, 42 uracil DNA glycosylase, 43 DNA pol III χ subunit, 44 PriA helicase, 45 and RecO. 46 To better address the direct interaction between RecJ and SSB, in vitro coimmunoprecipitation studies using antibodies against the two proteins were carried out.…”

Structure of the RNA Polymerase Domain of E. coli Primase

“…Although DnaG is capable of synthesizing 60nucleotide-long primers in vitro (3), this primer length is restrained to 11 ( 21) nucleotides in the context of the replisome (7). During lagging-strand synthesis of the E. coli genome, DnaG proteins must transcribe -2000 to 3000 RNA primers at a rate of about one primer per second (8).…”

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

“…One relies on the selective recruitment of DnaG to a specific site of action. For example, Escherichia coli DnaG interacts with both the replicative helicase, DnaB (Griep and Lokey, 1996; Marians, 1992; Tougu et al, 1994; Wu et al, 1992a), and the single-stranded DNA binding protein, SSB (Sun and Godson, 1996; Sun et al, 1994), interactions that allow DnaG to interface with distinct replisomal processes (Heller and Marians, 2006; Tougu and Marians, 1996a). A second strategy involves the localized control of primer synthesis.…”