Ch

Replication

FIGURE 5.21 Methylation after DNA Duplication

Dam methylase recognizes GATC palindromic sites and methylates them prior to DNA strand separation. The complementary sequences are synthesized during DNA replication but are not immediately methylated. Later, Dam methylase adds methyl groups to the newly synthesized sequences.

Hemi-methylated DNA

Hemi-methylated DNA

Methylation

(Dam methylase)

used to initiate chromosome replication. Hemi-methylated DNA binds to the cell membrane, helped by SeqA (sequestration protein), whereas fully methylated DNA does not.

The preceding implies that methylation and membrane binding are necessary controlling factors for initiation of replication. This is by no means the whole story, as dam mutants of E. coli, lacking the ability to add methyl groups, are viable and grow quite well. Thus, an origin with no methylation at all must be functional. Mutants lacking SeqA protein initiate replication more frequently than normal but are also viable. The factor(s) that control membrane binding are still obscure, as is the timing mechanism that oversees how long the origin is bound to the membrane and hidden from Dam methylase.

DNA replication finishes at special sites in the terminus region of the chromosome.

Chromosome Replication Terminates at terC

Replication finishes when the two replication forks meet at the terminus of the chromosome. This region is surrounded by several Ter sites that prevent further movement of replication forks (Fig. 5.22). The Ter sites act asymmetrically. TerC, TerB and TerF prevent clockwise movement of forks and TerA, TerD and TerE prevent counterclockwise movement. Since replication proceeds in two directions in prokary-otes, the meeting of the two replication forks is prevented by sets of Ter proteins at the termination region. The two innermost sites (TerA and TerC) are most frequently used and the outer sites presumably serve as back-ups in case a fork manages to slip sequestration protein (SeqA) Protein that binds the origin of replication, thereby delaying its methylation Ter site Site in the terminus region that blocks movement of a replication fork terminus Region on a chromosome where replication finishes

FIGURE 5.22 Termination of replication by Tus and Ter Sites

The circular bacterial chromosome has a termination region, or terminus, with several sites that stop replication forks moving clockwise (TerF, TerB and TerC) and counterclockwise (TerE, TerD and TerA).

FIGURE 5.22 Termination of replication by Tus and Ter Sites

The circular bacterial chromosome has a termination region, or terminus, with several sites that stop replication forks moving clockwise (TerF, TerB and TerC) and counterclockwise (TerE, TerD and TerA).

past TerA or TerC. For example, the clockwise replication fork might pass through TerE, TerD, and TerA sites with no problem, until it is halted by TerC.

The Ter sites have a 23 bp consensus sequence that binds Tus protein. This blocks the movement of the DnaB helicase and brings movement of the replication fork to a halt. Tus binds asymmetrically and stops movement from one direction only. It can be displaced from the DNA by a fork coming from the other direction. However, the whole terminus region of E. coli (including the gene for the Tus protein, which is located next to TerB) can be deleted without apparent ill effects. This implies that the replication forks do not need to meet at a Ter site and can terminate successfully wherever they collide.

If circular molecules of DNA become interlocked, specific enzymes are needed to untangle them.

Disentangling the Daughter Chromosomes

When a circular chromosome finishes replicating, the two new circles may be physically interlocked or catenated (see Ch. 4). Such catenanes must be separated so that each daughter cell receives a single chromosome upon cell division (Fig. 5.23). Decatenation of interlocked circles is carried out by topoisomerase IV. Although the terminology is confusing, Topo IV is actually a type II topoisomerase whose mode of action is similar to DNA gyrase. Topo IV is found behind the replication fork, where it untangles the newly formed DNA as replication proceeds. It can also decatenate finished DNA circles, both of chromosomes and plasmids.

A related problem sometimes results from recombination. The two growing circular chromosomes may recombine even during the process of replication. Each pair of crossovers or exchanges of genetic material may cause the growing circles to become interlocked. If there is an even number of crossovers, Topo IV can disentangle the circles and no harm is done. However, an odd number of crossovers will covalently join the two circles of DNA (Fig. 5.24). The covalent dimer must be separated by the crossover resolvase, XerCD, which uses the dif sites on the two chromosomes to introduce a final crossover. This gives, in effect, an even number of crossovers.

crossover resolvase Bacterial enzyme that separates covalently fused chromosomes dif site Site on bacterial chromosome used by crossover resolvase to separate covalently fused chromosomes Tus protein Bacterial protein that binds to Ter sites and blocks movement of replication forks

Disentangling the Daughter Chromosomes

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