Term
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Definition
| deoxy chain terminators lack a 3' hydroxyl and thus block DNA elongation |
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Term
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Definition
- replication chain terminators
- target viral replication because they are better substrates for viral polymerases
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Term
| what are molecular probes? |
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Definition
cross linking analogs are used to capture DNA-protein interactions BrdU labeled DNA can be visualized with α-BrdU antibodies |
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Term
| What are the properties of DNA polymerase? |
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Definition
- they cannot initiate a DNA chain; they can only elongate an existing one
- most are template directed; they use a template for sequence information
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Term
| In general, DNA polymerase requires: |
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Definition
- activated precursors in the form of dATP, dCTP, dGTP, or dTTP
- Mg+, as a catalytic co-factor
- A primer to be elongated
- a template to direct the selection a complementay nucleotide
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Term
| What is the kinetic rate of an E.coli DNA polymerase? |
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Definition
| replicates at a rate of about 1,000 bases/sec |
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Term
| How is replication started? |
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Definition
| it is initiated by primase |
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Term
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Definition
| a RNA polymerase that synthesizes a short RNA primer |
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Term
| Name the different DNA polymerases |
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Definition
Pol α Acts with primase to initiate replication Pol β DNA repair polymerase Pol γ Mitochondrial DNA polymerase Pol δ Major nuclear DNA polymerase Pol ζ Damage bypas polymerase |
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Term
| What are the steps of initiation? |
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Definition
- primase joins RNA nucleotides into a promer
- DNA polumerase adds DNA nucleotides to primer
- DNA polymerase sequences newly made DNA
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Term
| What is proofreading and how do cells go about doing it? |
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Definition
Many DNA polymerases contain a 3'-->5' exonuclease proofreading function that can backup and remove misincorporated nucleotides |
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Term
| What is the overall misincorporation rate? |
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Definition
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Term
| How does a cell handle a mismatch repair? |
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Definition
- if C is misincorporated, the it will block further elongation
- 3' to 5' exonuclease activity is attached to DNA polymerase
- chews back to create a base-paired 3'-OH and on the primer strand
- DNA polymerase continues the process of adding nucleotides to the base-paired 3'-OH end of the primer
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Term
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Definition
- it coordinates replication on both strands
- goes in 5'-->3', so the leading strand is synthesized continuously and lagging strand is synthesized decontinuously.
- each segment of lagging strand synthesis is initiated by primase
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Term
| How are leading and lagging strands coupled? |
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Definition
-they are coupled to a ring-shaped processivity clamp |
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Term
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Definition
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Term
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Definition
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Term
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Definition
seals nicks between lagging strand fragments |
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Term
Topoisomerase(type I and II) |
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Definition
type I-removes twists in front of the fork type II-untangles daughter double helices |
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Term
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Definition
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Term
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Definition
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Term
How does Topoisomerase work |
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Definition
- one end of the DNA helx cannot rotate relative to the other end
- DNA topoisomerase covalently attaches to a DNA phsophaste, thereby breaking a phosphodiester linkage in one DNA strand
- the two ends of the DNA double helix can rotate relative to each other, reliveing accumulated strain
- the two ends of the DNA double helix can now rotate relaive to each other, relieving acculated strain
- the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible
- spontaneous re-formation of the phospho-diester bond regnerates both the DNA helix and the DNA topoisomerase.
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Term
| how does the cell deal with the shortening strands |
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Definition
teleomerase-is an RNA template, DNA polymerase (a reverse trancriptase) that carries its own template and adds sequences to the end of chromosomes |
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Term
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Definition
the combination of the teleomere sequence and the proteins that bind them also protect chromosome ends from degredation and fusion. |
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Term
| End replication strategies to deal with "shortening ends" |
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Definition
Circular chromosomes in telomerase-/- yeast and cancer cells, recombination copies sequence from one end to another in fruit flies, transposons add sequence to chromsome ends |
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Term
How does telomerase behave? |
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Definition
- telomerase binds
- telomerase extends 3'end (RNA templated DNA synthesis)
- completion of lagging strand by DNA polymerase (DNA-templated DNA synthesis)
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Term
| why does the "end of replication problem" arise? |
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Definition
- RNA priming and DNA synthesis
- removal of primers and filling gaps with DNA where a 3' end is available
- further rounds of replication
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Term
The regulation of DNA replication |
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Definition
- S phase: replication is confined to specific time during cell cycle, called S for synthesis
- Allows cells to make nucleotides, polymerases, etc in one conc burst
- Initiation is the rate limiting step in replication; is it therefore the primary elongated step
- entry in to S phase is the major cell-cycle commitment point in the cell cycle
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Term
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Definition
starts at a specific site, an origin of replication in eukaryotes, replication initiates at specific locations along the chromosomes, spaced 10 to 100s of kbs apart (1000s in humans) -however, they are degenerate in sequence and difficult to recognize
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Term
Eukaryotic Origin Replication |
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Definition
origins are bound by 6-protein ORC (origin of replication complex) |
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Term
what factors does ORC recruit |
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Definition
recruits Cdc6 which recruits Mcm complex (helicase) |
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Term
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Definition
| binds to site and after recruitment of Cdc6, gets phosphorylated which causes the initiation of replication |
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Term
2D gel analysis of replication |
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Definition
1st dimension: separates mostly on size (M.W.) in low percentate agarose 2nd dimension: separates mostly on shape in high percentage agarose with EtBr |
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Term
| Assays to see if DNA is replicating |
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Definition
-do 2D gel -density shift/CsCl gradients to isolate replicate sequences, and hybridization -quantitative microarrays -labeled replicated DNA with BrdU, analyze with BrdU antibodies |
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Term
| What structures are these representing?[image] |
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Definition
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Term
What does the 2D gel represent? |
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Definition
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