S M Ee

Chromosome duplication - 40 min Cell division - 20 min

60 min

Chromosome duplication - 40 min

Chromosome duplication - 40 min

Cell division - 20 min

Cell division - 20 min ^^

40 min

FIGURE 5.26 Cell Division and Chromosome Replication

40 min

FIGURE 5.26 Cell Division and Chromosome Replication

A) Cell division in more than 60 minutes allows for a time gap after cell division and the start of the next DNA replication. B) Cell division in 60 minutes requires 20 minutes for replication and 40 minutes for completion of cell division. C) Cell division in less than 60 minutes requires that a new cycle of replication be initiated before the last replication is completed.

chromosome always takes 40 minutes and the time from termination of replication to completion of cell division takes 20 minutes. If the generation time is less than 60 minutes, one or more rounds of chromosome replication must overlap. This means that a new cycle of replication may start before the previous one has finished. Cells in rapidly dividing cultures of bacteria therefore contain multiple but incomplete copies of the chromosome. If the generation time is longer than 60 minutes, there is a gap between division of the cell and initiation of the next round of chromosome replication (Fig. 5.26).

Nucleic acid molecules that survive and divide must have both an origin of replication and be circular (or have ends that are protected).

The Concept of the Replicon

A replicon is any DNA (or RNA) molecule that is capable of surviving and replicating itself inside a cell. A replicon must possess an origin of replication where replication is initiated. A replicon must also be an intact, "complete" molecule of DNA (or RNA) with ends that are protected from being attacked by a cell's defense system.

replicon Molecule of DNA or RNA that contains an origin of replication and can self-replicate

FIGURE 5.27 The 5-End is Potentially Lost in Replication of Linear DNA

When an RNA primer is removed after initiating a strand of linear DNA the gap cannot be filled by DNA as there is no upstream 3' hydroxyl to accept nucleotides. Thus, linear DNA would be shortened during each replication cycle.

End of linear chromosome i

Primer (RNA)

Dna replication r

Gap after primer removal

New DNA

Extra DNA molecules, known as plasmids, are found in many bacteria. They are usually circular and much smaller than chromosomes.

Although chromosomes are clearly replicons, they are by no means the only ones.Virus genomes replicate when inside their host cell. Consequently, their nucleic acid qualifies as a replicon. Since some virus genomes consist of RNA, this means that the definition of replicon must include both DNA and RNA.

In prokaryotes, replicons are usually closed circles of DNA that have no ends. In most bacteria, linear molecules of DNA are degraded by exonucleases. These are enzymes that degrade nucleic acids one nucleotide at a time, starting from one end or the other. Consequently, linear segments of DNA that enter a bacterial cell during conjugation or transformation (see Ch. 18) will eventually be degraded. If some of the genetic information carried on such DNA is to survive, it must recombine onto a circular replicon, similar in form to bacterial chromosome.

Despite this, a few bacteria contain linear chromosomes. These have a variety of individual adaptations to protect the ends from endonucleases. Borrelia burgdorferi, which causes Lyme disease, has hairpin sequences at the ends of its linear chromosome. Streptomyces lividans, a soil organism, has proteins covalently attached to the ends of its DNA.

Plasmids are another group of replicons. They are extra self-replicating molecules of DNA that are not necessary for survival of the host cell (see Ch. 16). Plasmids are usually circular, although linear plasmids occur in Borrelia and Streptomyces, the same bacteria that contain linear chromosomes. Circular replicons are occasionally found in eukaryotic cells, including plasmids such as the 2|m circle of yeast. Mitochondria and chloroplasts also contain their own genomes, or replicons, which are circular molecules of self-replicating DNA.

Replicating Linear DNA in Eukaryotes

Replicating a linear molecule of DNA requires certain adaptations. Since DNA polymerases can only elongate, not initiate, new strands of DNA must be initiated with an RNA primer. Since synthesis always proceeds from 5' to 3', one of these RNA primers must be located right at the 5'-end of each new strand when replicating linear DNA (Fig. 5.27). When this terminal RNA primer is removed, it cannot be replaced with DNA, as there is no strand for DNA polymerase to elongate. If nothing was done to overcome this problem, the molecule of DNA would grow shorter, by the length of an average RNA primer, with each round of replication. Circular prokaryotic DNA molecules do not have ends and so do not have this problem.

exonuclease Enzyme that cleaves nucleic acid molecules at the end and usually removes just a single nucleotide Lyme disease Infection caused by Borrelia burgdorferii and transmitted by ticks plasmid Accessory molecule of nucleic acid capable of self-replication. Does not normally carry genes needed for existence of host cell. Usually consists of double stranded circular DNA but occasional plasmids that are linear or made of RNA exist.

Replicating Linear DNA in Eukaryotes 127

One solution to the problem of initiation of replication in linear DNA is to use a protein primer at the ends. We normally think of DNA polymerase as only being able to extend nucleic acid chains. However, strictly speaking, DNA polymerase can add nucleotides only to a free —OH group. Although this free —OH group is normally furnished by DNA itself, or by an RNA primer, some DNA polymerases can add nucleotides to a free —OH group on specific proteins. This solution is used by several viruses and for the linear plasmids and chromosomes of Streptomyces.

OH group-used for priming

Tenninalj5 3 ^

protein ^

OH (P Terminal

OH 5' protein

FIGURE 5.28 Protein Primers for the Ends of Linear DNA

The terminal proteins of some viruses and plasmids bind to the 5'-end of linear DNA. These proteins have special OH groups that allow priming of DNA synthesis. The result is complete replication of the linear DNA, without "end-shortening."

Eukaryotic DNA is linear and needs special structures, the telomeres, to protect its ends.

Eukaryotes have adapted to the problem of replicating linear DNA by developing structures known as telomeres that are located at the ends of their chromosomes. Telomeres consist of multiple tandem repeats (from 20 to several hundred) of a short sequence, usually of six bases (TTAGGG, in vertebrates including humans). During protein primer Protein used instead of RNA as a primer for DNA synthesis in some bacteria and viruses telomere Specific repetitive sequence of DNA found at the end of linear eukaryotic chromosomes

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