Biochemistry

Lecture 18: DNA Replication & Repair

Assigned reading in Campbell: Chapter 8.1-8.7

Take the Review Quiz on Lecture 18 concepts.

Handout: Enzymes & Proteins that act on DNA is a worksheet to accompany this lecture.
10.16.00 Answers for the above worksheet.

Please note: The shaded sections of these revised lecture notes will not be covered this term.
10.13.00

1. DNA Replication: an overview.

Much of the complexity found in DNA replication (compared, say to transcription) derives from the requirements in all organisms for:

• high accuracy in duplicating the primary genetic information;
• high speed and processivity, so as to complete a copy of the genome in less than one cell generation (cell division time).

1. DNA polymerases can add nucleotides only to a pre-existing chain.
2. All DNA (and RNA) polymerases synthesize chains from 5' --> 3'.
3. DNA synthesis occurs at replication forks.

a) Synthesis is bidirectional from a replication origin (or origins).

b) Replication forks are highly organized structures in cells (DNA synthesis "machines").

1. DNA gyrase introduces negative superhelical turns (endergonic).
2. The reaction is coupled to ATP hydrolysis (net exergonic).

1. The leading strand is synthesized continuously, 5' --> 3'.
2. The lagging strand is synthesized in segments using RNA as primers (and as always, only in the 5' --> 3' direction).

1. Synthesized on the lagging strand template by DNA primase.
2. Synthesized by RNA polymerase on the leading strand during replication initiation at the origin.

1. Polymerization requires: a template, a primer strand, dNTPs, and Mg²⁺.
2. The reaction produces: an extended primer and PP_i.
3. Pol I edits its own errors and those made by others (e.g. other enzymes, and other DNA damaging events).

   a) 3' --> 5' exonuclease removes incorporation errors using a second active site (editing or proofreading). Products: dNMPs.

   b) 5' --> 3' exonuclease activity resides on a separate domain; it removes nucleotides in lengths from 1 to 10 nucleotides.

4. The 5' --> 3' exonuclease is essential for cell viability.

   a) this third active site removes damaged DNA starting from a "nick" (cleavage of the phosphodiester backbone, resulting in a 3'-OH and a 5'-phosphate).

   b) it also removes RNA from 5' ends of Okasaki fragments.

   c) "nick translation" results from the combined reactions of 5' --> 3' polymerization and 5' --> 3' exonuclease activity.

1. Pol III can only fill in gaps in double strand DNA.
2. Pol III has 10 subunits that assemble for replication (and rather easily disassemble during purification).

   a) the e subunit does the 3' --> 5' editing.

   b) the b subunit is the essential processivity factor (Campbell, Fig. 8.7).

3. Pol III is present in only ~20 copies/cell.

1. DnaB protein unwinds the parental helix (using ATP hydrolysis).
2. The unwound single-stranded DNA is coated with SSB (ssDNA binding protein).
3. DNA ligase repairs single-strand nicks, i.e. it catalyzes phosphodiester bond formation using ATP (or NAD⁺) as energy donor. The "Okazaki fragments" that result from lagging strand synthesis are joined by DNA ligase.

1. Replication initiates at the oriC site.

   a) DnaA protein is required along with HU.

   b) About 45 bp of DNA are melted near the origin.

   c) DnaB and DnaC insert into the melted open complex to form a "prepriming complex" with the release of DnaC.

   d) Assembly of accessory proteins follows.

   e) Pol III holoenzyme initiates synthesis on the leading and lagging strands.

The two replication forks are closely associated during replication of the chromosome (See Fig. 8.10 in Campbell.). Recent studies show that this DNA replisome complex is found at the center of the cell. This is also the location where specific cell division proteins assemble prior to the cell division event.
Thus, the current picture of E. coli chromosome replication is one in which the DNA synthesis and cell division "machines" are organized into coordinated replication "factories".
2. Initiation of replication is coordinated with cell division; the mechanism(s) are largely unknown.
3. Termination of Replication.

1. Balanced levels of dNTP's.
2. Two-stage nucleotide incorporation.

   a) Watson-Crick base pairing (to the template base) and stacking (on the primer terminus).

   b) hydrolytic editing of errors (3' --> 5' exonuclease).

   The incorporation accuracy is about 1 error/10⁶ nucleotides.
3. Repair enzymes keep DNA under constant surveillance.

1. DNA Pol a replicates the lagging strand.
2. DNA Pol d replicates the leading strand (with the PCNA processivity factor).
3. Multiple replication origins are required.

   a) multiple origins allow S phase to be short in duration.

   b) clusters of replication "machines" are organized into many DNA synthesis "factories". This higher-order organization is very similar to that seen in E. coli, except that there are many more factories in eukaryotic cells during S phase.

1. Retroviruses have a single stranded RNA genome that is converted to double stranded DNA in three steps:

   a) RNA is the template for RT-catalyzed synthesis of an RNA-DNA duplex.

   b) The RNA is degraded by the RNaseH activity of the viral RT.

   c) The DNA is copied (by host cell enzymes) to produce double stranded DNA. In this form, the retroviral genome can be inserted into the cellular chromosomes.

2. Inhibitors of HIV-1 RT that terminate a primer strand.

   a) dideoxy nucleotide analogues: ddI & ddC.

   b) similar mechanism of action: AZT & stavudine.

3. RT lacks a proofreading 3' --> 5' exonuclease.

   a) "error-prone replication" leads to mutations in all HIV-1 genes.

   b) the rapidly changing genome that results, is a difficult moving target to hit with therapeutic drugs.

1. Pyrimidine dimers are split by photolyase.

   a) the thymine dimer is "flipped out" of the helix into the enzyme active site for repair.

   b) reduction of the cyclobutane moiety requires light and FADH.

2. Alkylated nucleotides (e.g. 0⁶-alkylguanine) are repaired by "suicide enzymes". Each repair event irreversibly inactivates the repair enzyme by transferring the alkyl group on guanine to its own active site residue. In a sense, these enzymes operate as single-use reagents, and not as cycling catalysts.

1. UvrABC endonuclease cleaves damaged DNA on either side of a lesion thereby producing a short gap.
2. Pol I fills in the gap; DNA ligase completes the repair job.

We will not have time to consider the following topic this term. Nevertheless, as a Campbell-like "Biochemical Connection" to molecular genetics, the idea that mutation rates have been selected in evolution is a fascinating hypothesis. Replication accuracy varies among organisms. The finished replication accuracy is plotted vs. genome size. Both axes are logarithmic to accomodate the scale. The fact that these data fall on a line, shows that there is a constant mutation rate/genome/generation from the smallest virus (phage M13) to the eukaryotes (yeast and N. crassa). The average value obtained from these six examples is 0.0034 mutations/ genome/generation. Variation of this magnitude among species strongly suggests that the mutation rate has been selected in evolution. In other words, the balance between the speed and accuracy of DNA replication in any organism is an optimization that has been adjusted by natural selection. (The data are from Drake, J. et al. (1998) "Rates of spontaneous mutation" Genetics 148 1667-1686.)

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