Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 25 March

© R. Paselk 2006
 
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DNA Replication, cont. - Polymerases

Eukaryotic Polymerases and DNA Synthesis, cont.

Polymerase alpha was covered last time, let's next look at Polymerase del

Polymerase del 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 beta2 sliding clamp of prokaryotes, the homotrimeric protein forming a ring round the ssDNA and holding the polymerase in place.

Polymerase epsilon is similar to polymerase del, 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 beta has an unknown function, but is thought to be a repair enzyme, while Polymerase gamma is the mitochondrial DNA polymerase.

Chromosome Replication

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.

Telomere Replication

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 Repair

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:

  1. Excision and replacement of damaged DNA. Two basic systems of excision repair:
    1. Base excision: repairs single chemically modified bases by first removal of the base by DNA glycosylase, leaving the phosphosribosyl backbone intact, followed by a multi-enzyme, multi-step process which excises the ribose, cleaves the backbone and removes a number of additional residues, followed by repair of the resulting gap by polymerase and ligase.
    2. Nucleotide excision: repairs larger regions of DNA damage than base excision. In this system the excision repair system cleaves the backbone in two places spanning the damaged DNA, removes the DNA (12-29 nucleotides), followed by repair of the resulting gap by polymerase and ligase. This repair system is damaged in victims of Xeroderma pigmentosum, resulting in extreme sensitivity to sunlight damage to DNA and consequent lesions etc.
  2. Reversal of chemical changes in DNA.
    1. Mismatch repair: replaces incorrect bases inserted during DNA replication with the proper bases using polymerase and ligase.
    2. Photoreactivation of pyrimidine dimers: uses light energy to break UV induced bonds between, for example, thymine dimers, and restore the original bases.

Pathway Diagrams

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Last modified 26 March 2009