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INFORMATION METABOLISM

Requires a template

Replication

Transcription Translation

Each process consists of 3 distinct

subprocesses: initiation, chain

elongation and termination

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GENETIC TERMINOLOGY

Genome

Genetic map: linkage map or physical map

Phenotype/genotype

Allele

Marker

Copy numeber

Plasmid Wild-type

Reversion/ suppression (second-sitereversion)

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EARLY INSIGHTS INTO DNA

REPLICATION

Semiconservative nature of replication

Sequential nature of replication

Origin and direction of replication

Units of replication- initiation of replication is controlled by asmall cluster of genetic elements

-replicon

- a single chromosome is not always asingle replicon

-DNA replication is strongly controlled atthe level of initiation

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Quantitative Parameters of

Replicati

on

E.coli Human

DNA content,

nucleotide pairs/cell

3.9 x 106 109

Rate of replication forkprogression

30 m/min 3 m/min

DNA replication rate 850 nucleotides/sec per

fork

60-90 nucleotides/sec

per fork

Number of replication

origin per cell

1 103 104

Hours required to

complete geneome

replication

0.67 8

Hours to complete one

cell division

0.33 24

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OVERVIEW OF DNA REPLICATION

How does the coordination between celldivision and DNA replication occur?

How are the replication origins recognizedby enzymes?

What is the energy source for duplexstrand unwinding?

How many proteins must function to carryout process?

How are the activities of these proteinscoordinated?

Is there an additional fidelity-enhancingmechanism involved?

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DNA POLYMERASE

POLYMERASE I

- most abundunt

-has polymerase and nuclease activity-3 exonuclease serves as a

proofreading function

-5 exonuclease excise primers from the

lagging strand and repair DNA

(damaged by radiation or chemicals)

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DNA POLYMERASE

POLYMERASE II and III

-pol III has a major role of nucleotide

incorporation during elongation

-pol II participates in DNA repair

synthesis

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GENERAL STRUCTURE AND

MECHANISM

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DNA POLYMERASE III

HOLOENZYME

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CLAMP and CLAMP LOADER

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Uracil-DNA N-Glycosylase

DNA polymerases readily acceptsdeoxyuridine triphosphate as a substrate

Removes dUMP residues

Hydrolytically cleaves the glycosidicbond between N-1 uracil and C-1deoxyribose yielding an apyrimidinic site

Apyrimidinic endonuclease recognizesthis site and cleaves the phosphodiesterbond on the 5 side of the deoxyribosemoiety

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INITIATION OF DNA REPLICATION

What are the site-specific DNA-protein

interactions that trigger initiation?

How do proteins act after binding to

origin sequence?

How is the process controlled?

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REQUIREMENTS FOR INITIATION

A nucleotide sequence that specifically

binds initiation proteins

A mechanism that generates a primer

terminus to which nucleotides can be

added by DNA polymerase

In general, these origins include

repeated sequences of either identicalor opposite polarity

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REQUIREMENTS FOR INITIATION

2 ways to generate a primer terminus:

1. Nicking a strand of the parental duplex

to expose the 3 hydroxyl terminus

2. Unwinding the parental duplex and

synthesizing an RNA primer to expose

a 3 hydroxyl ribonucleotide terminus

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INITIATION ofE. coliDNA

REPLICATION

Starts at the oric

245 bp long

Contains 4 repeats of 9 bp sequencethat binds an initiation protein DnaA

Contains 3 repeats of 13 bp sequence

that is rich in A and T

Also contains binding sites for proteins

HU and IHF that facilitate DNA bending

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MITOCHONDRIAL DNA

REPLICATION

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MITOCHONDRIAL DNA

REPLICATION

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REPLICATION OF LINEAR

GENOMES

Phage T4 and T7

Bacteriophage 29

Eukaryotes

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Phage T4

and T7

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Bacteriophage

29

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Eukaryotic DNA

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Reverse Transcription