The Orf protein can partially substitute for RecORF in Red-mediated recombination between the bacterial chromosome and short linear dsDNA molecules, in promoting the replication of a pSC101-derived plasmid, and in recovery of a cell from UV-induced damage. These findings are superficially inconsistent with those of a previous study, which suggested a more limited capability of Orf to substitute for RecORF (34
). However, in the previous study, orf
was expressed from a plasmid. As noted by the investigators, in a recBC sbcB sbcC
background, RecORF and Orf both might be expected to influence the tendency of a plasmid to produce potentially lethal linear multimers (10
). Moreover, there is indirect evidence that plasmid linear multimers can inhibit recombination, perhaps by competing for a limited supply of recombination proteins (24
The ability of Orf to substitute for RecORF has suggested all along that Orf may perform the same mechanistic function as RecORF (33
). The term “RecORF” (also appearing in the literature as “RecFOR”) recognizes that the three polypeptide products of the recF
, and recR
genes are thought to function as a physically interacting complex in the same step(s) in recombination and gap repair (7
). Genetic (44
) and biochemical (4
) evidence indicates that the key RecORF-promoted step is the loading of RecA protein onto single-stranded DNA (ssDNA), displacing the E. coli
single-stranded DNA-binding protein (SSB).
That RecORF's main role in recombination is, in effect, to assist RecA was indicated by the isolation of srf
(suppressor of recF
) alleles of recA
). The protein encoded by one such allele, RecA803, has been shown to compete more effectively than wild-type RecA with SSB for binding to ssDNA (16
). Both a recA803 recBC sbcB sbcC
strain and a Δ(recC ptr recB recD
)::Ptac gam bet exo pae cI
strain are capable of recombining efficiently in the absence of RecORF. This observation led to the prediction that RecA803 might do for the Δ(recC ptr recB recD
)::Ptac gam bet exo pae cI
cell what Orf does: promote efficient RecORF-independent recombination. The inability of RecA803 to promote RecORF-independent recombination via the Red pathway (Table ) raised the question of why RecA803 needs RecORF to load it onto ssDNA, given that RecA803 can displace SSB by itself. One interesting possible answer is that for Red recombination, RecA needs to displace the Beta protein, not SSB, from ssDNA.
The idea that a dsDNA end acted on by Red is converted to a Beta protein-coated 3′-ended ssDNA is consistent with the enzymology of λ exonuclease (Exo) (12
), the interaction between Exo and Beta (32
), and the DNA-binding properties of Beta (8
). The idea that RecA has to displace Beta to act on this DNA end has implications for our understanding of the Red pathway.
According to the model for Red recombination developed by Stahl and coworkers (37
), the Red pathway is best understood as having two branches, with one proceeding via strand annealing and the other proceeding via strand invasion. The strand invasion mechanism requires RecA, while strand annealing is RecA independent. RecA-promoted strand invasion is necessary when only one of the two recombining partners has a free end and the other does not, as is the case in the recombination event monitored in this study involving the lac
dsDNA segment and the (uncut) bacterial chromosome.
The inference, outlined above, that RecA must displace Beta to form recombinants via the strand invasion pathway suggests that the progression of a double-strand-break repair-recombination event down one or the other branch of the Red pathway is kinetically regulated by the type of partner available to the linear DNA species (Fig. ). The sequence of events in Red recombination, according to this view, would be as follows. (i) An end produced by a double-strand cut to a chromosome is acted on by the Exo-Beta complex. Exo digests the 5′-ended strand, and at the same time, deposits the Beta protein on the exposed 3′-ended single strand in a way which is analogous to the deposition of RecA protein on ssDNA by RecBCD after the interaction of the latter with a χ site (1
). (ii) If a complementary ssDNA is present in the cell, Beta promotes annealing, forming a recombinant. (iii) If a complementary ssDNA is not present and a dsDNA with shared sequences is present, recombination will still take place, but only after RecORF (or possibly Orf) removes Beta from the 3′-ended ssDNA and replaces it with RecA.
FIG. 2. Roles of recombination proteins in the early steps of the Red pathway. DNA intermediates are drawn according to the Stahl model (37). (A) A free dsDNA end is acted on by the Exo-β complex, leaving a 3′-overhanging ssDNA species coated (more ...)
The idea of sequential and mutually exclusive actions by Beta and RecA suggests that strand annealing is the primary Red pathway while strand invasion is a salvage pathway, only taking place when strand annealing is blocked. In the salvage pathway, perhaps the only role of Red is to convert a dsDNA end into a 3′ overhang, and a large number of additional cellular recombination proteins may be needed to make a recombinant.
The sequential Beta-RecA hypothesis raises the following question: why is Beta required at all in the strand invasion pathway? Beta is specifically required for Red activity in both strand annealing and strand invasion events (26
). Possibly, Beta modulates the exonuclease activity of Exo, which otherwise would destroy recombination intermediates.
The surprising finding of this study was that Orf complements null mutations in ruvAB
. In the study that originally characterized Orf functions, this activity could not have been detected, as the recombination event under investigation was Ruv independent (33
). The Ruv-complementing activity was observable in a cell which was mutated only in ruvAB
, not in genes for other known repair-recombination functions (Fig. ), implying that it is not a peculiarity of the highly engineered Δ(recC ptr recB recD
)::Ptac gam bet exo pae cI
genetic background in which most of the experiments were done.
The RuvA, RuvB, and RuvC proteins have been shown to act in concert to resolve branched DNA molecules which model recombination intermediates (46
). RuvB is a helicase which drives branch migration and which is targeted to Holliday junctions by the RuvA protein (40
). RuvC is an endonuclease (resolvase) which specifically cleaves Holliday junctions at symmetrically related strands (3
). The phage λ-encoded Rap protein, which is also a junction-targeted endonuclease (36
), can, like Orf, partially substitute for RuvC, but not for RuvAB or other E. coli
recombination proteins (31
The pleiotropy of Orf action suggests that Orf does not directly replace the proteins whose functions it renders nearly unnecessary. It is readily conceivable that Orf might have the same functional activity as RecORF. If a single amino acid substitution in RecA, turning it into RecA803, is almost all that is needed to dispense with RecORF, there is no obvious reason why the same effect could not be achieved by even the small Orf protein, which has a monomer molecular mass of 16.6 kDa. It is harder to imagine that Orf could have the same enzymatic activities as RuvABC and is nearly inconceivable that it could mimic both RecORF and RuvABC.
One way in which Orf might indirectly suppress the phenotypes of strains lacking RecORF or RuvABC components is by inducing the expression of a set of cryptic genes which have RecORF- and RuvABC-like activities. Indeed, ruv
mutations are known to be suppressed by mutational activation of the cryptic rusA
). The ability of Orf to suppress a ruvC
mutation is rusA
independent, as it occurs in a genetic background from which rusA
and all the cryptic prophage genes in its vicinity have been deleted (Table ) (9
), but the possibility that Orf activates some other set of genes cannot be ruled out.
A second way in which Orf might work is by modifying some other protein or multiprotein complex, making it RecBCD-like, i.e., capable of carrying out moderately efficient recombination in the absence of RecORF or RuvABC (see references 14
for reviews). This hypothetical mechanism of Orf action is constrained by three observations, as follows. (i) In the Δ(recC ptr recB recD
)::Ptac gam bet exo pae cI
background, recombination and repair are independent of RecORF and RuvABC but are still highly dependent upon Red, despite the presence of Orf (Table ). It follows from this observation that there is no Orf-modified protein complex in the cell, other than Exo-Beta, which can operate on dsDNA ends to promote efficient exchanges, and also that RecG cannot be the Orf target. (ii) Suppressing effects of Orf are also seen in cells in which RecBCD is present (Table ). Therefore, having both RecBCD and Orf gives cells capabilities beyond those which result from having RecBCD without Orf. (iii) Orf does not suppress a recQ
mutant (Table ), leaving open the possibility that RecQ might be the Orf target. This possibility is made unlikely, however, by the observation that the slow growth phenotype conferred by Orf is not suppressed by a recQ
null mutation, as would be expected if Orf acted only by modifying RecQ; the recQ
mutation itself does not noticeably affect the growth rate (data not shown).
A third way in which Orf might work is by modulating the activity of SSB. This SSB modulation hypothesis provides a ready explanation for how Orf could help RecA displace SSB from ssDNA. Orf-modulated SSB may also help RecA to compete with Beta for ssDNA binding. The expression level-dependent growth slowing or stopping activity of Orf can also be readily understood as an effect of its interfering to various degrees with SSB's function in DNA replication (see reference 20
for a review). It is not obvious how SSB modulation could account for Orf's RuvABC-bypassing activity. The essential role of SSB in replication makes a precise evaluation of its role in recombination difficult. However, there is evidence indicating that SSB has an essential role in recombination as well as in replication (6
). The current understanding of recombination mechanisms permits speculation that the resolution of recombination intermediates can occur by a number of different pathways (13
), but at least in a Δ(recC ptr recB recD
)::Ptac gam bet exo pae cI
strain, only RuvABC is fast enough to resolve them in such a way as to form recombinants efficiently. If RuvABC is not present, perhaps other fast processes resolve the intermediates without forming recombinants. If SSB has a critical role in any of these other processes, then modulation of its activity by Orf might slow them down or accelerate still other recombinant-forming processes.