Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 11 March	© R. Paselk 2002
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DNA Replication 2

Mitotic Cell Cycle

In eukaryotes DNA replication in dividing cells takes place during a specific phase of the cell cycle, as noted below.

1. Mitosis (nuclear division with conservation of chromosome number) and cytokinesis (cell division) occur during the brief Mitotic (M) phase of the cell cycle. Mitosis is divided into five subphases in which the chromosomes are arranged and separated:
   1. Prophase
   2. Prometaphase
   3. Metaphase
   4. Anaphase
   5. Telophase and Cytokinesis
2. Mitosis is followed by the much longer Interphase in which cell growth and chromosome replication occur. Interphase is also divided into subphases. The biosynthesis of proteins, cell organelles etc., with the exception of DNA, occurs during all of the subphases.
   1. G₁ (fist gap) phase. This is generally the longest phase of the cell cycle. Early in G₁ proteins bind to the ORC (origin replication complex) to make a pre-replication complex (pre-RC). A variety of other proteins required for replication are also synthesized and/or form complexes during this phase to allow later DNA replication in the next phase.
   2. S phase is the only phase where DNA is synthesized and chromosomes duplicated.
   3. G₂ (second gap) phase follows S phase. It is a relatively short phase in which additional growth takes place in preparation for the next cycle of mitosis.

Eukaryotic Polymerases and DNA Synthesis

Our studies will focus on the occurrences in S phase, beginning with DNA replication. As with prokaryotes, eukaryotes have a number of different polymerases specialized for different aspects of DNA replication and repair, as summarized in the table below:

Polymerase Organelle Other Activities Structure Processivity  Fidelity Inhibition a nucleus Primase 250 kD: tetramer of 180 kD core; 70 kD ?; 60 kD & 50 kD primase subs. Low, about 200 bp High Aphidicolin & N-Ethylmaleimide b nucleus 36-38 kD Low Low Dideoxy NTP's g mitochondrian 3'-5' exonuclease 160-300 kD: 125 kD core; 35 kD; 47 kD High High Dideoxy NTP's & N-Ethylmaleimide d nucleus 3'-5' exonuclease 170 kD: dimer with 125 kD core, 50 kD sub which associates with PCNA. High High Aphidicolin & N-Ethylmaleimide, weak by Dideoxy NTP's e nucleus 3'-5' exonuclease 256 kD: 215 kD core; 55 kD High High Aphidicolin & N-Ethylmaleimide, weak by Dideoxy NTP's

The specific functions of these polymerases were established using specific inhibitors, combinations of which could block various polymerases while allowing others to continue.

Polymerase a is a nuclear polymerase which participates in the replication of the chromosome, functioning in the initiation of DNA replication on the lagging strand. When provided with a ssDNA template it first synthesizes an RNA primer of about 10 nucleotides, then adds up to 20 or so deoxynucleotides. Note the very low processivity makes proofreading unnecessary.

• In lagging strand synthesis, polymerase a first adds a primer and a stretch of about 20 deoxynucleotides. Synthesis is then switched (see below) to polymerase d (or e), and additional deoxynucleotides are added. Priming happens frequently, occurring about every 50 nucleotides. The primer is removed, except for the last nucleotide, by RNase HI removes all but the last nucleotide, which is then hydrolyzed off by a complex called FEN1/RTH1. The gap is filled by polymerase, then the resulting Okazaki fragment is joined to the growing strand by DNA ligase.

Polymerase d 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 b₂ sliding clamp of prokaryotes, the homotrimeric protein forming a ring round the ssDNA and holding the polymerase in place.

• In leading strand synthesis, polymerase a first adds primer and a stretch of deoxynucleotides. Replication factor C (RFC), the eukaryote analog of the prokaryote g factor, then removes polymerase a and aids the assembly of PCNA (polymerase switching). Polymerase d then binds to the PCNA trimer and synthesizes the leading strand until a stop signal is reached.

Polymerase e is similar to polymerase d, except that it does not require PCNA. It appears to be used for repair, and possibly for lagging strand synthesis in conjunction with polymerase a.

Polymerase b has an unknown function, but is thought to be a repair enzyme, while Polymerase g 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.

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Last modified 20 March 2002