Bio 105
DNA Replication
1/28/12
BASIC FEATURES OF DNA REPLICATION IN VIVO
DNA Synthesis ≠ DNA Replication (DNA Synthesis occurs also during DNA repair and recombination.)
Essential Features of Cellular DNA Replication
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Semiconservative (i.e. single-strand template)
Fixed Origins [Unique (bacterial) or Multiple (eukaryotic)]
Bidirectional (2 replication forks per origin)
3' OH polynucleotide primer required (for DNA Polymerases but not RNA Polymerases)
Priming by RNA
Strand elongation in 5' → 3' direction (for DNA and RNA Polymerases)
(d) N5'TP precursors
Many proteins required
SEMICONSERVATIVE REPLICATION
Review Meselson-Stahl Experiment thoroughly.
For E. coli DNA Polymerase II you do not need to memorize all the subunit designations
(Greek letters) shown in Fig. 10.15/10.26
UNWINDING DNA WITH HELICASES, DNA-BINDING PROTEINS, AND TOPOISOMERASES
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Bio 105
DNA Replication
1/28/12
THE REPLICATION APPARATUS: PREPRIMING PROTEINS, PRIMOSOMES, AND REPLISOMES
Shortlist of Proteins Required at Replication Fork (in E. coli)
PROTEIN
Helicases (Rep and Helicase II)
FUNCTION
Cooperate to unwind the DNA ahead of the replication fork
at rates approaching 1,000 bp/second. Requires ATP
hydrolysis.
Rep moves 3' → 5' on leading strand template.
Helicase II moves 5' → 3' on lagging strand template.
Topoisomerase (Gyrase)
Relaxes positive supercoiling introduced by unwinding.
Single-Strand DNA Binding Protein Stabilizes single strands.
(SSB)
Primase
RNA primers (10 b) for Okazaki fragments
(As part of “primosome”)
5' → 3' DNA template-dependent RNA polymerase
DNA Polymerase III
leading and lagging strand DNA synthesis
[7 subunits]
5' → 3' DNA template-dependent DNA polymerase
DNA Polymerase I
("Kornberg Polymerase”)
proofreading
3' → 5' ss DNA exonuclease
removal of RNA primers
5' → 3' ss RNA exonuclease
DNA synthesis in gaps created by primer removal
also repair/recombination)
5' → 3' DNA template-dependent DNA polymerase
DNA Ligase
proofreading
3' → 5' ss DNA exonuclease
Seals single strand nicks remaining between adjacent
Okazaki fragments in discontinuous replication.
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Bio 105
DNA Replication
1/28/12
UNIQUE ASPECTS OF EUKARYOTIC CHROMOSOME REPLICATION
Genomic DNA replication is limited to the S phase of the eukaryotic cell cycle.
Replication forks move about 2.5 kb per minute
Each chromosome may have several hundred to several thousand replication origins
Huberman and Riggs 3H-thymidine labeling and autoradiography. Fig. 10.29/10.31
"REPLICON": A segment of genomic DNA replicated from a single origin (and having two
termini).
Each replicon in a eukaryotic chromosome may be 30-300 kb.
Telomerase: Replication of Chromosome Termini
Telomerase is a “reverse transcriptase”.
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