Hisao Masai and Ken-ichi Arai.

Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108, Japan

Received 01/17/96; Accepted 02/17/96; On-line 03/01/96

6.1 dnaT (protein i)

In order to examine the physiological function of the phiX174-type primosome in replication of the E. coli chromosome, we undertook molecular genetic analyses of primosomal proteins for the phiX174-type primosome. First, we isolated the gene for protein i, which was mapped next to dnaC (32, 33). Further genetic analyses indicated that protein i is encoded by dnaT. dnaT and dnaC constitute an operon and are cotranscribed together with the p18 gene downstream of dnaC from a promoter present upstream of dnaT (33). dnaT was originally isolated as being defective in induction of iSDR (34) . This result suggests for the first time that the phiX174-type primosome may function in iSDR.

6.2 PriA (protein n')

n' protein recognizes the n'-pas and triggers assembly of the phiX174-type primosome. We isolated the gene for this protein and designated it priA (4, 35). priA is a previously unknown gene located at 88.7 min on the E. coli chromosome. PriA protein belongs to the DEXH-type RNA/DNA helicase family (Figure 2A) and it does have intrinsic ATPase and DNA helicase activities which are specifically stimulated by n'-pas.

.......... .......... .MpvahVALP VPLPRtFDYl lPeGMt.vka
.......... .......... .MkIVrVALa VPLPRlFDYf VPddvs.lqi
maglpalppg srelfpedah AepVVaVlLP lPLagayDYk VPaGMarpav

GcRVRVPFGk QqERIGIVvs vsdASElPln eLKAvvevLD sePvFThsvW
GmRVlVPFGt Qk.RvaIVad fptkSdvPed kLKAilqpLD laPlFTpiyW
GtlVRVPlGr reE.IGvVwg .agAgEtPpe rLKplig.fp ecPplpaplr

rlLlWAAdYY hhPiGDVLFh ALPilLRqGr pAa.NApmWy wfateQgqAv
dwLhWAAnYY qaglGDVLFq ALPVkLRnGe sAvkNdrtfw RitdagknAl
afidWvAaYt vqPpGaVLrm ALsV..paal eApppAlgWr RpsagQraAg

d........l nsLKRSPKQQ qALAaLrqgk iwr....Dqv RtleFndAal
k........q GeLKRSkKQa eALqyLsetd lek....gnn ...dFssAiw
qraegqgplp GgarlSPgrQ rvLAvLddhp glpfagaDla ReaavgpAvv

qALrKKGLcd ...lasetpe fsdWrtNyav ..sgeRLrLN teQAtAvGai
sALkaKGfiE eiTiqtnpls wqqrlgNnPi vnaenRLtLN kQQAlAfsql
aAmaKaGLlE avT......r sneWspqaPd adrpgpLlsa dQQAaAdGlr

hsaadtFSaW LLaGVTGSGK TEVYLsvlEn vLaqGKQALV mVPEIGLTPQ
lfhsg.FnvW LLdGVTGSGK TEiYLqyIEE iLksGKQvLV LVPEIGLTPQ
taldqgFSgl LLeGVTGSGK TEVYfeaIaE tLrrGrQALV LlPEIaLaaQ

TiaRFreRFN ApveVLHSgL nDseRLsAWl kAknGeAAIV IGTRSALFTP
TvqRFkvRFN veidVLHSnL tDtqRLyvWd rArsGqsAIV IGTRSALFTq

wprRFadRFg AapvqwHSqm gaaaRrrAWr avalGrApvV vGaRSALFlP

FkNLGvIviD EEHDSSYKQQ EGWRYHARDL AVyRAhseqI PiiLGSATPa
FsNLGaIIlD EEHDSSYKQQ dsWRYHARDL AiVlAqklnI svlmGSATPS
ypdLGlIIvD EEHDSafkQe EGvpYnARDm AVVRArlggf PavLaSATPS

LETlcNVQQk KYRlLrLtRR AGNArpaiqh VLDLKgQkvQ ..........
LEsInNVQnG KYqHLVLskR AGNstalrhf ViDLKnQniQ ..........
LETIeNarQG rYRHLVLpRR hGgAempeit lLDLrrappQ kwlptdfagp

.......... .......... .......... .......... .....aGLaP
.......... .......... .......... .......... .....nGLSk
ggseglaapg gandeaeeqk apppsptasp sptaspspma rrarlgwLSP

aLITRMrqHL qAdNQViLFL NRRGFAPaLL CHdCGWIAeC PRCdhyYTlH
PLleRMkaHL ekGNQVLLFL NRRGFAPvLL CHeCGWIAqC PhCekpYTyH
PLITaveetL aAGeQVLLFL NRRGyAPltL CrsCGhrlkC PRCtawlveH

QaQhhLRCHH CdsQrPvPRQ CPsCGSTH.L VPvGLGTEQL EqTLApLFPg
QhQnvLRCHH CGaQktIPRQ CgdCGSTH.L VttGLGTEQL EETLktLFPh
rrdgrLRCHH CGyQqPIPet CPaCGvadsL aPcGpGvErL aEeaAhrFPk

vpisRIDRDT TSRKGaLEqq LaevhrGgar ILIGTQMLAK GHHFPdVTLV
ysvaRIDRDs TSRKGkLEgy LedIqqGksq ILIGTQMLAK GHHFPnVTLV
armdvaasDT vtgpkeaaal atrIanhdid liIGTQimAK GHHFPliTLV

ALlDVDgALF SaDFRsaERf AQLYtQVAGR AGRAgKQGEV VLQTHhPeHP
ALVnVDsALF SlDFRAeERl AQLYiQVAGR AGRAdKQGEV VLQTHyPdHP
gvVDgDlgLt ggDlRAsERt hQLlhQVAGR AGRAerpGrV liQTvdPgHP

LLqTLLykGY dAFAEqrLAE RrmMqLPPwT shVivrAedh nnqhAplfLQ
LLtTLLanGY qAFAketLql RhsMgLPPFT fqalikAqar hSdlAencLs
vmeaLasgdp alFlEveaAE RqalamPPFg rlValvisge dSar....vQ

QlrnlilSsp ladekLwVLG PVPAlapKrg GRwRWQiLLQ HPSRVrLQhi
QiAdffqSkq itG..LqmLG PmPAPfsKka GqyRWQLLLQ HPSRmtLQka
avAaalgraa pmGpgLdVLG PVPAPlamlr GRhRhrLLLk aargVkvQpv

ingtLaLint iPdsrkVKwv LDVDPIegz.
lReyqq..ae leknsqVrli LDVDPqdlsz
vRhwLsLvsi pPg...VKvq vDVDPIsfl.

Figure 2: Schematic representation of the structure of E. coli PriA protein and comparison of its primary structure with possible homologues form other bacterial species. A Six conserved sequence motifs for DNA/RNA helicases and the clusters of cysteine residues for a possible Zinc-finger structure are shown by gray and black boxes, respectively. The numbers indicate the amino acid number from the N terminus. B:The amino acid sequences of the E. coli PriA and those of two candidate homologues from Haemophilus influenzae and Rhodospirillum rubrum are aligned for maximum identity. The amino acid residues identical in at least two genes are shown with capital letters. Sequences from Haemophilus (Fleischmann et al., Science 269, 496 [1995]) and Rhodospirillum (Falk et al., Biochem. J. 228, 391 [1985]) are under the accession number HIU32718 and X02499, respectively, in Genbank. Homology with the Rhodospirillum rubrum gene was previously reported (4).

An additional feature of the structure of PriA protein is the four repeats of CXXC (C, cysteine) which are inserted between the helicase conserved motifs. This region is likely to constitute a Zinc finger-like structure. Mutagenesis study indicated the cysteine residues are essential for priA function (36). Search of data base lead to identification of likely homologues of priA from two species, one from Haemophilus influenzae and the other from a photosynthetic bacteria, Rhodospirillum rubrum (Figure 2B). Identity (I) and similarity (S) are as follows:

• E. coli (733 aa) vs. Haemophilus (710 aa) : I, 52%; S, 71 %

• E. coli (733 aa) vs. Rhodospirillum (812 aa): I, 39%; S, 58%

The conserved motifs for helicases and CXXC sequences are precisely conserved among all the three proteins, indicating the importance of those sequences for priA function. Inactivation of the chromosomal copy of the priA gene by insertion of a kanamycine resistance gene (priA1::kan) can be tolerated for viability of the E. coli cells (37, 38), but priA1::kan cells grow extremely poorly and do not grow on rich medium (27). They are highly sensitive to DNA damaging agents such as UV and mytomycin C and exhibit filamentous morphology (39). Requirement of protein i for iSDR prompted us to examine involvement of PriA protein for iSDR. We measured iSDR in the priA1::kan strain, and discovered that iSDR was completely lost in the mutant strain (27). It was recovered by introduction of a plasmid carrying the wild-type priA gene. We constructed a double-mutant of rnhA224 and priA1::kan and measured cSDR. cSDR was also completely wiped out in this strain (27). Furthermore, cSDR-dependent growth of rnhA224 and dnaA(ts) double mutant at 42°C was also lost by priA1::kan mutation.These results demonstrate that PriA protein is essential for both iSDR and cSDR. They, in conjunction with requirement of dnaT for iSDR, strongly suggest that the phiX174-type primosome is responsible for replication forks during the course of SDR. A possible additional role of PriA protein in chromosomal replication could be "reloading" of replication complexes which may fall off the forks during the course of their propagation along the DNA template more than 2000 kb in length, although this possibility has not been experimentally tested.