DNA Replication and Telomeres Flashcards
What direction is DNA replication?
bi-directionally away from origins
Where is the DNA double-helix opened?
at the origin of replication and unwound to form replication forks
What are exposed at replication forks?
single stranded DNA is exposed and DNA synthesis can occur
What are the continuous and discontinuous strands called?
the leading strand and lagging strand
What is the function of helicase?
begins bi-directional DNA unwinding creating two forks
What does DNA polymerase need?
a primer
What is DNA primase?
a specialized RNA polymerase that synthesizes RNA primers for DNA replication
Does RNA polymerase need primers?
do not need primers to begin RNA synthesis (same as transcription)
What is a primer?
a short (<10 base) RNA strand complementary to the template
What are the three steps of DNA replication?
- sliding clamp is recruited to primer
- DNA polymerase is recruited to sliding clamp
- All DNA synthesis occurs in 5’ to 3’ direction
What are Okazaki fragments?
nascent lagging strands (~0.2 kb in eukaryotes/ ~2kb in prokaryotes)
When does termination occur?
when two different forks meet, when the fork reaches the end of a linear chromosome, or when polymerase meets the previously replicated strand (as above)
What happens to RNA primers after termination?
removed and replaced with DNA
What are the main polymerases in DNA replication for bacteria?
polymerase III
What are the main polymerases in DNA replication for eukaryotes?
polymerase δ and ε
What is proofreading?
polymerases are highly conserved and composed of multiple subunits that have different functions
What is processivity?
DNA polymerases attach to DNA for long stretched before dissociation
After each nucleotide is added, DNA polymerase has the potential to _
fall off the DNA
What is proccessive synthesis?
replicative polymerases stay attached for many 1000s of nucleotides
What is distributive synthesis?
DNA polymerases involved in repair, only add a few nucleotides before falling off
What is tautomerization?
the isomerization of each base between its tautomeric forms
What causes improper incorporation?
the less common tautomer can pair with the wrong bases
What happens to misshapen DNA?
moves from the polymerase active site to a second enzymatic site on the protein that contains 3’ to 5’ exonuclease
What is the function of proofreading exonuclease?
removes the termine 3’ ase, thus restoring the correct geometry of the primer-template junction, which can move back to the polymerase site
What are the chances for incorrect nucleotide polymerization?
~10^-5
What are the chances for incorrent nucleotide after moving to a second enzymatic site?
~10^-7
What are chances for incorrect nucleotide after Mismatch Repair?
10^-9
The active sites of polymerases are _
highly conserved
How are polymerases grouped?
- group according to the evolutionary lineage of the rest of the protein
- in groups A, C, D, X, and Y
What suggests the early divergence of polymerase groups?
are more similar within groups than within organisms
What catalyzes unwinding?
catalyzed by ATP-hydrolysis-driven DNA helicases, which open up the helix and travel along the DNA, continuously unwinding it at the replication form
What direction does bacterial helicase move?
5’ to 3’ along the lagging strand template
What direction does eukaryotic move?
moves 3’ to 5’ along the leading strands
What does the crystal structure of helicase and DNA show?
a single strand of DNA within a hexamer of helicase subunits
How many subunits does E. coli helicase, DnaB, have?
six subunits
What is the helicase in euakroyres and archea called?
MCM complex and is formed of different, but related subunits MCM2-7
What makes copying by polymerase difficult?
single stranded DNA can form secondary structures that would make copying by polymerase difficult
What is the function of single-stranded binding proteins?
single-stranded binding proteins (SSA in bacteria and replication protein A, in eukaryotes) bind to the displaced single stranded DNA
SSB proteins keep _ and interact with the _
open, DNA synthesis machinery
What happens as DNA is unwound?
torsional stress is introduced into the DNA - strand separation at the fork results in overwinding ahead of the fork
What do torsional stresses lead to?
changes in DNA supercoiling - overwinding makes the DNA ahead of the for more difficult to unwind
What is the function of topoisomerases?
resolve the overwinding by transiently breaking DNA and allowing supercoils ro relax
What is the function of Type IA and IB topoisomerases?
break one of the two DNA strands and do not require ATP
What is the function of Type II topoisomerases?
use atp to separate two tangled helices by cutting both. strands of one helix and passing the other helix through the gap, ligates the two ends
What is the function of Type I topoisomerase?
cleaves one DNA strand and becomes covalently bonded to one end through phospho-tyrosine bond
What happens to the uncut strand after Topo I cleaves one DNA strand?
passed through the cut strands (Type Ia), or rotation occurs around the phosphodiester bond of the uncleaved strand (Type Ib), to relive tension (pos. or neg)
What ligates the two DNA ends?
topo I
What is the function of Topo II?
separates two tangled helices by cutting both strands of one helix and passing the other helix through the gap; ligates two ends
What is the high processivity of replicative DNA polymerase due to?
the sliding clamp that keeps it tethered to DNA
Both _ and _ are highly conserved in bacteria, archaea, and eukaryotes
clamp and clamp loader
What is a sliding clamp?
a ring shape with 35 A hole that encloses the DNA - it is highly stable and stays associated with DNA once loaded
What is a bacterial sliding clamp?
the beta protein; in eukaryotes it is PCNA
What is an E.coli ring?
a homodimer of two proteins with three similar domains; in the pCNA three proteins of two similar domains come together
How does the clamp bind DNA polymerase?
through an 8 amino acid motif
What is the mechanism of clamp loading (6)?
- the clamp loader has a low affinity for the sliding clamp until the loader is bound to ATP
- the clamp loader binds to the sliding clamp, forming a spiral shape
- the clamp loader-sliding clamp complex has a high affinity with the primer-template junction
- binding of the complex to the primer-template junction stimulates ATPase activity, which closes the sliding clamp and releases the clamp loader
- sliding clamp remains associated with DNA
- the clamp loader must be recharged with ATP in order to repeat the cycle
What happens during polymerase switching (2)
- after the polymerase alpha subunit of the primase complex makes a short stretch of DNA, replicative polymerases take over
- after the clamp loader releases, replicative polymerase binds to the sliding clamp in the position where the loader was previously bound
How often does polymerase switching occur?
every time a new strand is started
How is replicative polymerase recruited?
by the sliding clamp
What are joined after synthesis?
okazaki fragments - okazaki fragment maturation
What happens to DNA polymerase III in E. coli?
dissociates from the DNA when it reaches the next RNA primer. The sliding clamp remains attached
What does DNA polymerase I have?
a 5’ exonuclease that degrades RNA primer while its DNA polymerase activity fills in the gap with DNA. This results in “nick translation” because the nick has been moved along the DNA
What seals the DNA nick?
DNA ligase
