BICH/GENE 431 KNOWLEDGE OBJECTIVES
Chapter 8 – DNA Replication
origin of replication
replicons
unidirectional replication vs. bidirectional replication
leading strand vs. lagging strand / continuous vs. discontinuous replication
Overall, semidiscontinuous replication
Okazaki fragments and RNA primers
dNTPs; α, β, γ phosphates; incorporation of α phosphate into DNA chain
chemical reaction during DNA synthesis – pyrophosphate released and then hydrolyzed
to inorganic phosphates by pyrophosphatase
incorporation assay to monitor DNA synthesis
- either α-32P-dNTP or fluorescently-labeled T base in TTP as substrate
- separate synthesized DNA (bigger) from unincorporated dNTPs (smaller)
using filter binding or gel electrophoresis
- measure radioactivity or fluorescence incorporated into DNA
3D structures of several DNA polymerases known – model structural features after right
hand (palm, fingers, thumb)
processivity of DNA polymerases
fidelity of DNA polymerases
- approximate error rates in the cell, and by enzyme with/without proofreading
- kinetic control of fidelity
- proofreading activity: 3’ to 5’ exonuclease
many different DNA polymerase enzymes in E. coli and humans
Two major DNA pols in E. coli + 3 others
- DNA pol I: discovered first by Arthur Kornberg, most abundant, single subunit,
not very processive; 3 activities: 5’ to 3’ polymerase, 3’ to 5’ exonuclease
(proofreading), 5’ to 3’ exonuclease for endfilling after removal of Okazaki
fragments
- DNA pol III: major replicative enzyme, highly processive, multiple subunits to
be discussed later
Three major DNA pols in eukaryotes + many others
- Polα used for primer synthesis
- Polδ used for lagging strand synthesis
- Polε used for leading strand synthesis
Many other proteins, enzymes at the replication fork in addition to DNA polymerase
Primase catalyzes addition of RNA primers
DNA helicases unwind DNA
Single-stranded binding proteins (SSBs) keep DNA single-stranded after unwinding
Topoisomerases relax supercoils that are introduced into DNA because of unwinding
Know names of these proteins, enzymes in E. coli, humans (Table 8-1)
Process used to remove RNA primer and seal phosphodiester backbone after
completing new synthesis in E. coli:
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