Information Copying

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INFORMATION METABOLISM

� Requires a template

� Replication

� Transcription� Translation

� Each process consists of 3 distinct

subprocesses: initiation, chain

elongation and termination



GENETIC TERMINOLOGY

� Genome

� Genetic map: linkage map or physical map

� Phenotype/genotype

� Allele

� Marker

� Copy numeber

�

Plasmid� Wild-type

� Reversion/ suppression (second-sitereversion)



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



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



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?



DNA POLY MERASE

� 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)



DNA POLY MERASE

� POLYMERASE II and III

-pol III has a major role of nucleotide

incorporation during elongation

-pol II participates in DNA repair

synthesis



GENERAL STRUCTURE AND

MECHANISM



DNA POLY MERASE III

HOLOENZY ME



CLAMP and CLAMP LOADER



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 phosphodiester bond on the 5¶ side of the deoxyribosemoiety



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?



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



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





INITIATION of E. coli DNA

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







MITOCHONDRIAL DNA

REPLICATION



MITOCHONDRIAL DNA

REPLICATION



REPLICATION OF LINEAR

GENOMES

� Phage T4 and T7

� Bacteriophage 29

�

Eukaryotes



Phage T4

and T7





Bacteriophage

29





Eukaryotic DNA



Reverse Transcription

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