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?Issues to be resolved during DNA replication
- 1. mechanism must exist for DNA to unwind and be STABLE in open position: helicase and ssBP
- 2. Unwinding and synthesis create tension further down helix which needs to be reduced: topoisomerases
- 3. Primer needs to be synthesized for polymerization to start: primase
- 4. Both strands synthesized simultaneously, but in different manners (continuous w/leading and lagging): DNA polymerase III, replisome, okazaki fragments
- 5. RNA primer must be removed prior to completion of replication: DNA polymerase I
- 6. Gaps between strands must be filled: ligase
- 7. DNA needs to be proofread for errors: DNA polymerase I and III
How to help telomeres from shortening the DNA replicating strands
Telomerase: binds to the end GGTTT sequences with complementary bps, allowing the 3í end DNA template to NOT create the hairpin loop, thereby allowing the complementary strand to FINISH replication
DNA replication in prokaryotes
- circular shape molecule, up to few mil. bps
- SINGLE origin of replication
- replication BIdirectional
- Both strands replicated simultaneously
- meeting/terminating at ter sites, producing 2 circular molecules
- about 20min
DNA Replication in eukaryotes
- MULTIPLE LINEAR DNA molecules (1 per chromosome), each millions of bps
- Multiple ori's
- need special mechanisms to fully replicate ENDS of each molecule
- several hours
Process and Proteins involved
- 1)Helicases unstabalize the helix opening up the DNA
- 2) Single strand binding protiens hold the helix in the open position
- 3) Topoisomerases (DNA Gyrase) relieves tension from supercoiling ahead of the helix
- 4) Primase ñ Synthesizes and lays down an RNA primer so DNA polymerase can begin synthesizing DNA
- 5) DNA Polymerase III begins synthesizing new DNA in the 5í-3í Direction
- 6) Leading strand is direct but lagging strand done in small pieces ñ Okazaki fragments
- 7) DNA polymerase I ñ removes RNA primers and fills in space with DNA
- 8) DNA Ligase joins the phosphate backbones of the various fragments (fills in the nicks)
- 9) All DNA polymerase contain 3í-5í exonuclease activity allowing for proofreading and synthesizing of DNA in 5í-3í direction
◦ Single Stranded Binding Protein
SSBP (single stranded binding proteins) are homotetramers that bind to exposed ssDNA strands without covering the bases.
- ◦ The role of SSBP:
- 1. Protect ssDNA from degradation.
- 2. Control what proteins have access to the ssDNA.
- 3. Aid helicases by stabilizing the unwound ssDNA.
- 4. ssDNA can bond to itself forming hairpin helices. SSB prevent formation of the hairpin helices that impede synthesis by DNA polymerase. They exhibit cooperative binding on the ss-lagging strand of the DNA.
◦ Initiation in eukaryotes
- Initiation is the first step in translation
- 1. begins with METHIONINE
- 2. NO Shine-Dalgarno sequence
- 3. mRNA HAS 5' CAP
- 4. 12 Initiation factors
◦ Initiation in prokaryotes
1. N-formyl-methionine2. HAS Shine-Dalgarno sequence (Shows ribosomes where to start)3. Has Initiation factors 1, 2 & 3