|Lecture Notes: 25 March||
DNA Replication, cont. - Polymerases
Polymerase was covered last time, let's next look at Polymerase
Polymerase is the primary polymerase in eukaryotes, synthesizing the leading strand, and aiding in lagging strand synthesis. Note that its 3'-5' exonuclease activity enables it to proofread as it synthesizes, giving it a high fidelity. When associated with PCNA (proliferating cell nuclear antigen) via its 50 kD subunit it is essentially infinitely processive. PCNA is analogous to the 2 sliding clamp of prokaryotes, the homotrimeric protein forming a ring round the ssDNA and holding the polymerase in place.
Polymerase is similar to polymerase , except that it does not require PCNA. It appears to be used for repair, and possibly for lagging strand synthesis in conjunction with polymerase .
Polymerase has an unknown function, but is thought to be a repair enzyme, while Polymerase is the mitochondrial DNA polymerase.
Eukaryotes have multiple initiation sites on each chromosome. Each replication unit, or replicon, having 3-300 kb. The largest chromosome in D. melanogaster thus has about 6000 replicons.
Not all replicons are activated simultaneously. Rather, clusters of 20-80 adjacent replicons are activated throughout S phase until the entire chromosome is replicated.
Note that eukaryotic DNA replication is much slower than E. coli, with only 100-200 nucleotides in eukaryotic Okazaki fragments. However, the vast number of replication forks results in the entire genome being replicated in only about seven hours. Histones for packaging the DNA are synthesized concomitant with the DNA. The new histones going to the new DNA.
An additional replication problem for eukaryotes which is not shared with the eubacteria is the replication of chromosome ends. Eubacteria have circular chromosomes - there are no ends. This means that there is always a stretch of DNA which can be used as a template for a primer, regardless of strand direction etc.
For eukaryotes, on the other hand, one end of each strand will have the situation where there is no complementary strand before the replication start point to build a primer. This means that the first ten or so nucleotides of the replicating strand will be lost each replication cycle to the production of a primer. Eventually, no matter how much "junk" may reside at the end of a DNA strand, replication cycles will thus eat into the critical information containing DNA of an organism and it will cease replication and die (or go extinct).
Obviously this has not happened, so there must be a way around it. The secret is a special "reverse transcriptase" enzyme, telomerase, which can add additional nucleotides to a 3'-DNA strand end to replace those lost. It turns out that chromosome ends have many repetitions of a short sequence of bases. In vertebrates the repetitive sequence is TTAGGG. Telomerase is a ribonucleoprotein with an RNA strand containing a 9-30 nucleotide template sequence. The human telomerase has an RNA strand 450 nucleotides in length with a template sequence of -CCCUAA-. Telomerase can then use the 3'OH of DNA as a primer and its own template to a terminal -TTAGGG-OH sequence to the DNA. DNA polymerase can then use this sequence to add the complementary 3'-AATCCC-5' deoxynucleotide sequence, enabling another round of telomerase activity. Multiple rounds lead to an eventual cap of repetitive DNA 1-12 kbp long.
DNA is the only molecule which is repaired by the cell; other molecules such as protein, and RNA are simply discarded and replaced when damaged. DNA is constantly being damaged and thus must be repaired on a regular basis in order to assure the integrity of genetic information. Of course DNA is also repaired because it can be - it is the only molecule with built-in redundancy (due to the duplex helix) - which is also one reason it is the information archive molecule for life.
There are a couple of fundamental types of DNA repair:
Last modified 26 March 2009