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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 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)
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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
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GENERAL STRUCTURE AND
MECHANISM
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DNA POLY MERASE III
HOLOENZY ME
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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 phosphodiester bond 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 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
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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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