DnaA is targeted by other mechanism that inhibit rapid reinitiation atnewly synthesized

copies of oriC. As desribed above, only DnaA bound to ATP can direct initiation of
replication; however, this bound ATP is converted to ADP during the initiation process. Thus,
this process of directiing a round of replication initiation inactivates DnaA, preventing its
reuse. The process of exchancing the bound ADP for an ATP is a slow one, further
delayingthe accumulation of replication competent ATP bound DnaA. The process of
replicating nearby to bind ot oriC. There are more that 300 dnaA 90MER BINDING SITES
OUTSIDE of oriC (DNA also acts a transcripstional), and as they are replicated, this number
doubles. The increase in DnaA-binding sites acts to reduce the levels of available DnaA.
Together, these methods rapidly and dramatically reduce to abulity of E.coli to initiate
replication from new copies of oriC. Although these mechanisms prevent rapid reinitation,
this inhibition does not necessarily last until cell division is rate, the daughter copies of oric
must initatie replication prior to the completin of the previous round of replication. This is
cecause E.colli cells can divide every 20 minutes, but it takes more than 40 minutes to
replicate the E.coli genome. Thus, under rapid growth conditions E. Coli cells reinitiate
replication once and sometimes twice prior to the completion of previous rounds of
replication (BOX 8-7 Gambar 2). Even under such rapid growth conditions, initiation does
not occure more that once per round of cell division. Thus, for each round of cell division,
there is only one round of replication initiation from Oric.
Kotak 8-7. Gambar 2. Origins of replication reinitiate
replication prior to cell division in rapidly growing
cells. To allow the genome to be fully replicated prior
to each round of cell divisions, bacterial cells
frequently have to initiate DNA replication from their
single origin prior to the completions of cell division.
This is in contrast to eukaryotic cells, which do not
start chromosome segregation until all the

The complex of MutS and mismatch- containing DNA recruits MutL, a second protein
component of the repair system. MutL, in turn, activates MutH, an enzym that causes an
incision or nick on one strand near the site of the mismatch. Niking is followed by the action
of a specific helicase (UvrD) and one of three exonucleases (see below) . the helicase the
DNA, starting from the incision and moving in the directionof the site of the mismatch, and
the exonuclease progessively digest the displaced single strand, extending to and beyond the
site of the mismatched nucleotide. This action produces a single-strand ga, which is then

filled in by DNA polymerase III (Pol III) and sealed with DNA ligase. The overall effect is to
remove the mismatch and replace it with correctly base-paired nucleotide.
But how does the E.coli mismatch repair system know which of the thow mismatched
nucelotides to replace? If repair occured randomly, then half the time the error would become
pemanently establised in the DNA. The answer is that E. Coli tags the parental strand by
transient hemimethylation as we now describe.
The E.coli enzym dan ethylase methylase A residues on both strands of the sequences 5
GATC-3. The GATC sequence is widely distributed along the entire genome (occuring at
about once every 256 bp [44 ] ), and all of these sites are methylated by the Dam methylase.
When a replication fork passes throuhg DNA that is methylated at GATC sites on both
strands (fully methylated DNA), the resulting daughter DNA duplexes will be
hemimethylated (i.e, methylated on only the parental strand). Thus, for a few minutes, until
the Dam methylase catches up and methylase thr newly synthesized strand, daughter DNA
duplexes will methylated only on the strand that served as a template (Fig. 9-5a). Thus, the
newly synthesized strand is marked (it lacks a methyl group)and hence can be recognized
strand is marked ( it lack a methy group) and hence can be recognized as the strand for repair.
The MutH protein binds at such hemimethylated sites, but its endonuclease activity is
normally latent. Only when it is contacted by MutL and MuTS located at a nearby mismatch
(which is likely to be within a distance of a few hundred base pairs) does MutH become
activated as we described above. Just how this interaction takes place over distances of up to
several hundred base pairs is uncertain, but recent evidence indicates that the MutS-MutL
complex leaves the mismatch and moves along the DNA contour to reach MutH at the site of
he,imethylation. Once activated, MutH selectively nicks the unmethylated strand, so only
newly synthesuzed DNA in the vicinity of the mismatch removed and replaced (Gambar 95b). Methylation is therefore a memory device tha enables the E. Coli repair system to
retrieve the correct sequence from the parental strand if an error has been made during
replication.

Different exonucleases are used to remove single-stranded DNA between the nick created by
MutH and the mismatch, depending on whether MutH cuts DNA on the 5 or the 3 side of
the misincoporated nucleotide. If the DNA on the 5 or the 3 side of the misincoporated
nucleotide. If the DNA is cleaved on the 5 side of the mismatch, the exonuclease VII or
Rec], which degrades DNA in 5-> 3 direction, removes the stretch of DNA from the MutHinduced cut through the misincoporated nucleotide. Conversely, if the nick is on the 3 side