DNA Metabolism

• DNA replication: processes which DNA is being faithfully duplicated.

• DNA recombination: processes which the nucleotide sequence of DNA is being rearranged.

• DNA repair: processes which the structural integrity of DNA is being maintained.

Objectives of DNA replication• Basic mechanisms: (i) semiconservative, conservative, or rand

om dispersive, (ii) continuous,semidiscontinuous or discontinuous, (iii) unidirectional, bidirectional, or rolling circle.

• Enzymology: (i) identification of genes involved in replication , (ii) biochemical function of the protein products of these genes.

• Replicon: the unit of DNA replication. A single DNA molecule may consist of one replicon (eg., in prokaryotes) or many replicons (eg., in eukaryotes).

• Replication of any replicon may be separated into three phases: initiation, elongation and termination. The methods for identification of replication origin and the enzymes involved in each phase will be discussed.

• Regulation of DNA replication – how cells ensure each DNA is replicated only once per cell cycle.

Models of DNA replication

Prediction of experimental outcomes

DNA replication is semiconservative

DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cation such as Mg++, (v) synthesis is from 5’ to 3’.

Models of DNA chain elongation at replicating fork

Fig. 20.5

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)

Semidiscontinuous or discontinuous?

BioEssays 27:633-636 (2005)

Fig. 20.6

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

Fig. 20.7

RNA primers 10-12 nt long are used to synthesize Okazaki fragments

Fig. 20.8

Modes of DNA replication

• Bubbles (eyes) and Y structures.

• Theta mode (circular DNA).

• Displacement loop (D-loop).

• Rolling circle (Lariat or Sigma form)

Bubble or eyes.

Fig. 20.10

Theta mode.

Fig. 20.9

Rolling circle replication

Fig. 20.13

Fig. 20.14

Displacement loop

Fig. 13.11

Fig. 13.5

Directionality of DNA chain elongation

Fig. 20.11

Unidirectional replication of colicin E1 DNA

Fig. 20.12

Enzymology of DNA replication

• Identification of genes involved in replication: (i) isolation of conditional lethal mutants (eg., temperature-sensitive mutations) that affect DNA synthesis, (ii) map and clone the gene of interest.

• Biochemical function of the protein products of these genes.

Mutations

• A mutation is defined as any change in the nucleotide sequence in the DNA.

• Classes of mutations: (i) nucleotide displacement (base-pair substitutions), (ii) microinsertion and microdeletion (frameshift mutations), (iii) macroinsertion and macrodeletion, (iv) rearrangements (inversion, translocation etc.).

• Suppressor mutations, conditional lethal mutations, temperature-sensitive mutations.

Fig. 20.20

Fig. 20.22

Proteins involved in the initiation of replication

Proteins involved in the elongation of DNA

Initiation of Replication• Start of DNA chains: (i) RNA primer, (ii) terminal

protein primer, (iii) parental strand primer.• Identification of origins: (i) physical mapping: EM,

two-D gel electrophoresis etc., (ii) genetic mapping, (iii) functional mapping by DNA cloning.

• Chromosomal origins: (i) E. coli and other bacterial origins, (ii) origins without an initiator protein (ColE1 and T7), (iii) origins cleaved by initiator endonucleases, (iv) yeast autonomously replicating sequences (ARS).

• Initiation from the E. coli oriC.

Terminal protein may be used as primer to initiate replication

Fig. 13.13

Fig. 13.14

Fig. 13.15

Parental DNA strand as primer: Nicking by specific endonuclease to produce 3’-OH

Fig. 13.16

Fig. 13.18

Fig. 13.22

Fig. 13.23

Fig. 13.19

Two-dimensional gel electrophoresis to identify origin

Fig. 21.5

Fig. 21.6

Mapping of SV40 origin by EM

Fig. 21.3

Functional cloning of replication origin

Bacterial replication origins

The yeast origins of replication are contained within ARSs thatare composed of 4 important regions (A, B1, B2, and B3). An 11-bp (5-[T/A]TTTAPyPuTTT[T/A]-3’) consensus sequence is highly conserved in ARSs.

Fig. 21.7

Initiation from the E. coli oriC.

Fig. 21.1

Elongation at a replication fork

• Replication speed.

• Enzymes involved in DNA elongation at a replication fork and their functions.

• Model of simultaneous synthesis of both DNA strands by PolIII.

Fig. 21.8

Fig. 21.9

Proteins involved in the elongation of DNA

Elongation

DNA Polymerases

Processivity of DNA polymerase is determined at low enzyme concentration and in the presence of excessive substrates.

Holoenzyme consists of two cores, two subunits and one complex held together by a dimer of . So it is an asymmetric dimer.

Fig. 21.17

Fig. 21.16

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

Fig. 21.19

Fig. 21.20

Fig. 21.21

Fig. 21.23

Fig. 21.24

Fig. 21.25

Model for eukaryotic DNA replication

Termination of Replication

• Circular genomes: (i) termination sequences of E. coli, (ii) production of catenanes, (iii) decatenation by topoisomerase (TopIV in E. coli).

• Linear genomes: (i) end-replication problems of linear DNA, (ii) specialized structure in eukaryotic telomeres, (iii) maintenance of telomere length by telomerase and other mechanisms.

Fig.21.26

Fig. 21.27

TopIV participates in decatenation

Fig. 21.28

End-replication problem of linear DNA

Fig. 21.29

Formation of t loops in vitro.

Fig. 21.36

Regulation of DNA replication

• Control of initiation requires: (i) timing in the cell cycle, (ii) synchrony of initiation at multiple copies of oriC, and (iii) inhibition of immediate reinitiation.

• Processes required for initiation – protein and RNA synthesis, DNA methylation.

• DnaA level and timing of initiation.• DNA mehtylation in the regulation of initiation.• Regulation of ColE1 DNA replication.

Bacterial replication origins

Fig. 14.36

Fig. 14.37

Fig. 13.40

Fig. 13.41

Fig. 13.42

Fig. 13.43

A nucleus injected into a Xenopus egg can replicate only once

Licensing factor controls eukaryotic rereplication

Licensing factor consists of MCM proteins