Termination (DNA Replication)

The replication of genomic DNA can be separated into three stages: 

(1) Initiation, in which the replicative DNA helicase unwinds the origin of DNA replication. 

(2) Elongation, in which forks use semi-conservative DNA synthesis to duplicate the chromosome. 

(3) Termination, when converging replication forks meet, the process ends. Replication initiation is regulated in bacteria and eukaryotic cells so that genome duplication is limited to a single round of each cell cycle. Unlike initiation and elongation, which have been extensively researched, replication termination, particularly in eukaryotic cells, has received comparatively less attention. This is a significant gap in our understanding of genome duplication, especially since termination events are just as common as initiations, occurring 50,000 times in a normal S phase of mammalian cells.

Termination in Eukaryotes

In eukaryotic chromosomes, several replication sources exist, all of which begin replicating at the same time. On either side of the replication origin, a bubble of replicated DNA forms. The leading strand of one replication bubble eventually connects with the lagging strand of another bubble, and the lagging strand eventually connects with the 5′ end of the previous Okazaki fragment in the same bubble.

DNA polymerase, on the other hand, is unable to catalyse the formation of a phosphodiester link between the two segments of the new DNA strand, and hence fails to function. Nicks are detached regions of the sugar-phosphate backbone of a DNA strand that is otherwise fully duplicated.

The replication process does not end until all of the template nucleotides have been duplicated. DNA primers must be substituted for RNA primers, and nicks in the sugar-phosphate backbone must be repaired. 

FEN1 (flap endonuclease 1) and RNase H are two cellular enzymes that degrade RNA primers. FEN1 and RNase H are enzymes that remove RNA primers from the beginning of each leading strand and each Okazaki fragment, leaving gaps of unreplicated template DNA. Following the removal of the primers, a free-floating DNA polymerase lands at the 3′ end of the preceding DNA fragment and stretches the DNA over the gap. This, however, results in the formation of new nicks (unconnected sugar-phosphate backbone).

At each nick site, the enzyme ligase connects the sugar-phosphate backbones in the penultimate stage of DNA replication. Once all nicks have been patched by ligase and the daughter DNA molecule is complete, the new strand is one long continuous DNA strand.

Key Points

• When two replication forks meet on the same stretch of DNA, the following events happen, but not always in this order: All intervening DNA is unwound, any remaining gaps are closed and ligated, catenanes are eliminated, and replication proteins are emptied before the forks converge.

• In eukaryotes, the position of replication initiation sites determines the majority of termination sites. Termination in bacteria usually happens at a specific locus.

• Terminating replications can be a difficult task. The stalling of converging forks is required for SV40 replication termination, and bacterial termination is prone to induce re-replication. In eukaryotes, however, no fork stalling or re-replication has been reported after unperturbed termination.

• The formation of pre-catenanes, which are eliminated by type II topoisomerases, relieves topological stress that develops between converging forks. Fork convergence is required during bacterial and SV40 termination, but it is not required in eukaryotes.

• Following the convergence of the forks, a type II topoisomerase removes any residual catenanes. Gaps are easily filled in eukaryotes by extending the leading strands, although this process is less well characterised in bacteria and SV40.

• A specific replisome removal route has just been discovered in eukaryotes, and it operates late after termination after the DNA has been entirely copied. It’s unknown if microorganisms have a similar route.

• Termination is limited in circular bacterial chromosomes to a region known as the terminus area, which is located roughly opposite the replication origin.

• A replication fork trap is a stretch of DNA with an opposing arrangement of unidirectional replication terminator (Ter) sites that permits replication forks to enter but not escape.

• Terminator proteins attach to asymmetric DNA Ter sites to stop a replication fork from approaching the non-permissive side while allowing the fork to pass through to the permissive side.

• Over-replication of bacterial chromosomes and genomic instability come from failure to properly halt chromosome replication. 

CONCLUSION

Circular chromosomes are found in the majority of bacteria and archaea, and DNA replication starts at the origin of replication. Unwound double-stranded DNA forms two replication forks, which are activated by DNA polymerase complexes that progress each fork and copy the original strands in opposite directions away from the origin. The two forks meet and fuse, forming two independent double-stranded DNA molecules, which marks the end of DNA replication. This occurs at the terminus area, which is located diametrically opposite the origin, in the well-studied microorganisms Escherichia coli and Bacillus subtilis. Failure to properly stop chromosome replication can cause issues with genome function and stability, as well as DNA over-replication. Some archaea, on the other hand, have multi-origin chromosomes and don’t seem to regulate termination location explicitly.