CHAPTER 25 DNA Metabolism

DNA replication DNA repair DNA recombination

Key topics:

What is DNA Metabolism?

While functioning as a stable storage of genetic information, the

structure of DNA is far from static: A new copy of DNA is synthesized with high fidelity before each

cell division Errors that arise during or after DNA synthesis are constantly

checked for, and repairs are made Segments of DNA are rearranged either within a chromosome

or between two DNA molecules giving offspring a novel DNA

DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

DNA Metabolism

DNA replication: processes by which copies of DNA molecules are faithfully made.

DNA repair: processes by which the integrity of DNA are maintained.

DNA recombination: processes by which the DNA sequences are rearranged.

Map of the E. coli chromosome.

DNA Replication Is Semiconservative.

Replication Forks may Move Either Unidirectionally or Bidirectionally

Replication Begins at an Origin and Proceeds Bidirectionally in Many Bacteria Such as E. coli.

DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cathion such as Mg++.

DNA Elongation Chemistry

Parental DNA strand serves as a template Nucleotide triphosphates serve as substrates in stra

nd synthesis Hydroxyl at the 3’ end of growing chain makes a bo

nd to the -phosphorus of nucleotide Pyrophosphate is a good leaving group

DNA Synthesis Can’t be Continuously on Both Strands (because the DNA duplex is antiparallel and all DNA polymerases synthesize DNA in a 5’ to 3’ direction)

What is the source of primer used for lagging strand synthesis?

DNA Replication is Very Accurate Base selection by DNA polymerase is fairly accurate

(about 1 error per 104) Proofreading by the 3’ to 5’ exonuclease associated

with DNA polymerase improves the accuracy about 102 to 103-fold.

Mismatch repair system repairs any mismatched base pairs remaining after replication and further improves the accuracy.

An Example of Proofreading by the 3’ to 5’ Exonuclease of DNA Polymerase I of E. coli

Large (Klenow) fragment of DNA polymerase I retains polymerization and proofreading (3’ to 5’ exo)

DNA polymerase I has 5’ to 3’ exonuclease and can conduct Nick Translation

PolIII* consists of two cores, a clamp-loading complex ( complex) consisting of ’, and two additional proteins and . Holoenzyme is PolIII* plus subunits.

DNA polymerase III

The two subunits of PolIII form a circular clamp that surrounds DNA

DNA Replication requires many enzymes and protein factors Helicases: separation of DNA duplex. Topoisomerase: relieves topological stress Single-strand DNA binding proteins: stabilizes separ

ated DNA strands. Primase: synthesizes RNA primer. DNA Pol I: removes RNA in Okazaki fragments and

fills the gaps between Okazaki fragments. Ligase: seals nicks.

Replication of the E. coli chromosome

Initiation. Elongation. Termination.

Initiation begins at a fixed origin, called oriC, which consists of 245 bp bearing DNA sequences that are highly conserved among bacterial replication origins.

Model for initiation of replication at oriC.

Proteins involved in Elongation of DNA

Elongation: Synthesis of Okazaki fragments

Model for the synthesis of DNA on the leading and lagging strands by the asymmetric dimer of PolIII

Pol I can remove RNA primer and synthesize DNA to fill the gap

Termination: When the two opposing forks meet in a circular chromosome. Replication of the DNA separating the opposing forks generated catenanes, or interlinked circles.

Termination sequences and Tus (termination utilization substance) can arrest a replication fork

Replication in eukaryotic cells is more complex Contains many replicons. How is DNA replication initiated in each replicon is n

ot well understood. Yeast cells appears to employ ARS (autonomously replicating sequences) and ORC (origin recognition complex) to initiate replication.

More than one DNA polymerase are used to replicate DNA.

End-replication problem of linear DNA.

Assembly of a pre-replicative complex at a eukaryotic replication origin

The End Replication Problem of Linear DNA

DNA Damages

DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism.

DNA damage may be classified as: (i) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links.

DNA Repair and Mutations

Chemical reactions and some physical processes constantly damage genomic DNA

At the molecular level, damage usually involves changes in the structure of one of the strands

Vast majority are corrected by repair systems using the other strand as a template

Some base changes escape repair and the incorrect base serves as a template in replication

The daughter DNA carries a changed sequence in both strands; the DNA has been mutated

Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens

Ames test for mutagens (carcinogens)

Methylataion and Mismatch Repair

Model for Mismatch Repair

Base-Excision Repair

Nucleotide-Excision Repair in E. coli and Humans

Direct Repair: Photoreactivation by photolyase

Alkylation of DNA by alkylating agents

O6-methyl G, if not repaired, may produce a mutation

Direct Repair: Reversal of O6 methyl G to G by methyltransferase

Direct re

Direct repair of alkylated bases by AlkB.

Effect of DNA damage on replication: (i) coding lesions won’t interfere with replication but may produce mutation, (ii) non-coding lesions will interfere with replication and may lead to formation of daughter-strand gaps (DSG) or double-strand breaks (DSB).

DSG and DSB may be repaired by recombination process, to be discussed in the following section.

DNA repair and cancer

Defects in the genes encoding the proteins involved in nucleotide-excision repair, mismatch repair, and recombination repair have all been linked to human cancer.

Examples are: (i) xeroderma pigmentosum (or XP) patients with defects in nucleotide-excision repair, (ii) HNPCC (hereditary nonpoplyposis colon cancer) patients with defects in hMLH1 and hMSH2, and (3) breast cancer patients with inherited defects in BRCA1 and Brca2, which are known to interact with Rad 51 (the eukaryotic homolog of RecA) and therefore may have defective recombination repair.

Case Files: Case 11 (Breast cancer gene) and Case 13 (Fragile X syndrome).

DNA Recombination

Segments of DNA can rearrange their location within a chromosome from one chromosome to another

Such recombination is involved in many biological processes Repair of DNA Segregation of chromosomes during meiosis Enhancement of generic diversity

In sexually reproducing organism, recombination and mutations are two driving forces of evolution

Recombination of co-infecting viral genomes may enhance virulence and provide resistance to antivirals

DNA Recombination

Homologous recombination or generalized recombination.

Site-specific recombinataion. Transposition.

Pairing of homologous chromosomes and crossing-over in meiosis.

Recombination during meiosisis initiated by double-strand breaks.

Homologous recombination is catalyzed by enzymes The most well characterized recombination enzyme

s are derived from studies with E. coli cells. Presynapsis: helicase and/or nuclease to generate s

ingle-strand DNA with 3’-OH end (RecBCD). Synapsis: joint molecule formation to generate Holli

day juncture (RecA). Postsynapsis: branch migration and resolution of Ho

lliday juncture (RuvABC).

Helicase and nuclease activities of the RecBCD

RecA forms nucleoprotein filament on single-strand DNA

RecA filaments are extended and disassembled in the 5’ to 3’ direction

Filament assembly is assisted by RecFOR and RecX inhibits filament extension

RecA promotes joint molecule formation and strand exchange

Model for DNA strand exchange mediated by RecA

Models for recombinational DNA repair of stalled replication fork

Models for recombinational DNA repair

Site-specific Recombination: Bacteriophage lambda integration in E. coli

Effects of site-specific recombination on DNA structure

A site-specific recombination reaction (eg. catalyzed by Int of bacteriophage lambda)

XerCD site-specific recombinataion system can resolve dimer into monomer

Immunoglobulin Genes Are Assembled by V(D)J Recombination

Mechanism of V(D)J Recombination

Transposition

Transposition is mediated by transposable elements, or transposons.

Transposons of bacteria: IS (insertion sequences) contains only sequences required for transposition and proteins (transposases) that promote the process. Complex transposons contain genes in addition to those needed for transposition.

Transposition is characterized by duplication of direct repeats (5-9 bps) at target site.

Transposition, in some instances, may be mediated through a RNA intermediate.

Duplication of the DNA sequence at a target site when a transposon is inserted

Models for Direct and Replicative Transposition

Replicative transposition is meidatedby a cointegrate intermediate.

Fig. 23.6

Fig. 23.7