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4.3 DNA Replication and Repair When cells replicate through mitosis, it is important that each daughter cell has an exact copy of the parent cell’s DNA.
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4.3 DNA Replication and Repair • When cells replicate through mitosis, it is important that each daughter cell has an exact copy of the parent cell’s DNA. • Watson and Crick theorized that because the two strands of DNA were held together by hydrogen bonds, they would most likely replicate by separating (unzipping) and each strand could at as a template to create two identical DNA molecules. • DNA replicates semiconservatively. • An old strand matches with a new strand in the daughter cell. • Conservative replication would ‘conserve’ one original helix and create one entirely new helix.
Replication Vocabulary • DNA helicase: enzyme that unwinds double-helical DNA by disrupting H bonds • Anneal: the pairing of complementary strands of DNA through H bonding • SSB’s: single-stranded binding proteins, keep separated strands of DNA apart • DNA gyrase: the bacterial enzyme that relieves the tension produced by the unwinding of DNA during replication • Replication Fork:the region where the enzymes replicating a DNA molecule are bound to untwisted, single-stranded DNA • Replication Bubble: the region where 2 replication forks are in close proximity to each other, producing a bubble in the replicating DNA
REPLICATION of DNA • Replication begins when proteins bind to a replication origin. • DNA has multiple origins of replication • To expose a template strand, parent strands must be unwound and unzipped and kept separate. • Accomplished by DNA helicase • Breaks hydrogen bonds • Aided by single-stranded binding proteins (SSBs) • Bind to individual strands to prevent them from annealing.
When unwound, DNA is much too long to fit into a cell. • As a result, DNA is never fully unwound when replicating. • Replication proceeds towards the replication fork on one strand and away from the fork on the other. • When two replication forks are near one another, a replication bubble forms.
BUILDING THE COMPLIMENTARY STRAND • In prokaryotes, DNA polymerase I, II, and III are the main enzymes that function in DNA replication and repair. • Eukaryotes use 5 enzymes of DNA polymerase. • Enzyme in prokaryotes that builds complimentary strand using the template is DNA polymerase III • Only works under certain conditions.
DNA Polymerase III • Synthesizes DNA from 5` to 3`. • Adds deoxyribonucleosidetriphosphate to the 3` end of the elongating strand. • Requires and initial starting 3` end to commence elongation. • Cannot initiate a new complimentary strand by itself. • Must use a 10-60 base pair RNA primer • Synthesized by primase. • Primer is marked as `temporary` for ease of removal later
DNA polymerase III uses the energy derived from breaking the bond between the first and second phosphates to drive the condensation reaction that adds a complimentary nucleotide to the elongating strand. • Extra phosphates are recycled by the cell to build more nucleoside triphosphates. • Since DNA is synthesized from 5` to 3` direction and the template strands run antiparallel, only one strand can be built continuously.
The strand that uses the 3` to 5` direction is called the leading strand and is built towards the replication fork – continuous. • The other strand is synthesized in short fragments in the other direction and is known as the lagging strand – discontinuous. • Primers are continuously added and DNA polymerase III builds in short Okazaki fragments.
DNA polymerase I removes the primers and replaces them with appropriate deoxyribonucleotides. • DNA ligase joins the Okazaki fragments into one strand using phosphodiester bonds. The new double-stranded DNA automatically twists into a helix.
QUALITY CONTROL • DNA polymerase I and III `proofread` the newly synthesized strand. • When mistakes occur, either enzyme functions as an exonuclease. • The enzyme backtracks past the incorrectly paired nucleotide, removes it and continues adding nucleotides. • If the error is not repaired immediately, the mistake may be copied through subsequent passes. • If the error is missed by DNA polymerase I or III, there are several repair mechanisms that can fix it after replication.
