DNA Replication (contd.) Flashcards
RNase H function
- removes most of RNA primers’ nucleotides (except last one)
- auxiliary role for longer primers
DNA pol I function, directions(!)
- removes the last ribonucleotide in primers (that RNase H misses)
- fills gap with DNA (5’-3’)
- slow (10-20nt/sec)
- both exonuclease activities:
– 5’-3’ (removes ribonucleotides)
– 3’-5’ (proofreading)
what enzyme seals the gap between DNA fragments
DNA ligase
is DNA pol I:
RNA-dependent DNA pol (RNAP) or DNA-dependent DNA pol (DNAP)
DNAP! makes DNA from DNA template
basically replaces ribonucleotides with deoxyribonucleotides
endonuclease vs exonuclease
endonuclease cuts from the middle and cuts bonds between 2 bases (like inside the nucleotides)
exonuclease cuts at free ends only
DNA ligase function
- enzyme that links DNA fragments on lagging strand following primer removal and Pol I synthesis
- creates phosphodiester bond (b/w 3’OH + 5’PO4 of adjacent nucleotides)
- NEEDS ATP
topoisomerase role in DNA rep
- recognize + regulate supercoiling
- 2 types (I/II); type II (gyrase) NEEDS ATP
- introduces negative supercoils around oriC (necessary for initiation by DnaA)
- after helicase unwinds, positive supercoils form ahead of growing fork; TOPO II converts them into negative supercoils!
loop is growing; lagging core displaces ____ and finishes ____ ____
SSB
okazaki fragment
primase is positioned by ____ to make a RNA primer for lagging strand
helicase
replication fork for lagging strand (what happens after lagging core finishes an okazaki fragment)?
- primase, beta clamp, lagging core, gamma complex, RNase H, DNA pol I, ligase
- new primer made, primase released
- beta clamp released finished okazaki fragment from lagging core
- lagging core stays connected to leading core through tau subunit (dimer)
- gamma complex loads beta clamp to new primer
- lagging core binds to beta clamp, starts DNA synthesis
- RNase H and DNA pol I clean up ribonucleotides, DNA pol I fills gaps
- ligase connects fragments
what protein initiates by binding to oriC?
DnaA
what enzyme separates strands? what is its escort
helicase, DnaB (DnaC as escort)
what proteins keep strands apart during DNA rep?
single-strand binding proteins (SSB)
what enzyme is necessary to convert +ve supercoils to -ve?
topoisomerase II (gyrase, needs ATP)
what enzyme makes primers for both leading and lagging?
primase (DnaG, RNA polymerase)
lots of primers for lagging, one primer for leading
how does primase know where to make a primer?
primase positioned at right place through helicase - PRIMOSOME
what is a DNAPIII holoenzyme
the 2 cores; one for leading and one for lagging
why is DNAPIII processive?
beta clamp
what are the factors that load/unload beta clamp onto DNAPIII?
gamma complex
why is there synchronized replication of both strands?
2 core DNA pol are kept together by tau subunits
what enzymes work together to clean up RNA primers?
RNase H (except last one) + DNAPI (removes last one and fills in w/DNA)
what enzyme functions so newly made strands don’t have nicks?
DNA ligase
why is replicating bacterial genome called a theta structure?
the replication bubble looks like the theta symbol (bidirectionality), and end as two catenated loops
- forks meet at farthest point from oriC, following two termination forks (1 each)
how replication of circular DNA completes
since they are stuck together (2 loops)
- decatenation
- denaturation of unreplicated terminus, followed by supercoiling
- replication can complete before or after decatenation
- topo IV decatenates in vivo
- topo II can decatenate in vitro
euk vs prok DNA replication speed
euk: 50 bp/sec (more complex chromatin structure)
prok: 1000 bp/sec
how is euk diff from prok in DNA rep?
- speed (euk slower)
- multiple replicons
- euchromatin first
- “domino model” of origin activation during S phase
what is origin of replication in yeast?
autonomous replication sequence (ARS)
what is conserved in ARS?
A-domain of ARS (11bp consensus seq)
- mutation abolishes func
what recognizes euk origin (name)
eukaryotic origin recognition complex (ORC)
what is ORC made of?
6 proteins
- 5 subunits have winged-helix domain (WHD) and AAA+ domain (contains RecA fold and lid domains)
what are cyclins? what do they bind to? conc?
main proteins in cell cycle regulation
- bind to cyclin-dependent kinases (sets off cell cycle phase)
- diff ones are more active during diff cell stages
- gets degraded after triggering Cdk
licensing factor
- factor located in nucleus, necessary for replication
- Cdt1 - geminin, regulates lvls
- new rep needs new licensing factor to enter in nucleus (as old one is degraded)
- re-entry occurs in subsequence mitosis when nuclear membrane breaks
what are the polymerases for euk? their roles?
pol alpha - prime DNA synthesis (rep and repair)
pol delta - DNA rep of lagging strand (rep and repair)
pol epsilon - DNA rep of leading strand (rep and repair)
alpha pol = ?
primase
T/F: Pol I and III are in eukaryotes
FALSE, only prokaryotes
pol alpha’s interactions with pol epsilon and pol delta
pol alpha makes RNA primers,
- pol epsilon takes over for pol alpha on LEADING strand
- pol delta takes over for pol alpha on LAGGING strand
what is MCM?
helicase
what is cdc6?
helicase loader
what is FEN-1? coordinated by?
endo/exonuclease - removes primer
(RNase H)
coordinated by PCNA (sliding clamp)
what is PCNA?
sliding clamp
what is RFC?
clamp loader
euk DNA rep completion steps, coordinated by?
- FEN-1 removes primers from both strands (coordinated by PCNA)
- pol delta completes gaps on lagging strand (coordinated by PCNA and RFC)
- DNA ligase I seals gaps together on lagging strand
- nucleosomes are reassembled
why do we need telomeres and telomerases? what are they/their functions?
we lose bp with every replication
- telomeres contains repeating seq (TTAGGG) w/ protruded ss 3’end that can fold into loop, acts as a safety buffer
- telomerases - repairs and replicates telomeres
is telomerase RNA-dependent DNA pol or DNA-dependent DNA pol
RNA-dependent!
telomerase details
- ribonucleoprotein (protein-RNA complex)
- carries its own template RNA
- has reverse transcriptase activity
- tandem repeat (TTAGGG), results in 3’ overhang of lagging strand template
what is replicative cell senescence?
shortening chromosome ends, losing genetic info
- after many generations, descendent cells inherit defective chromosomes
- eventually stop dividing, stop cell cycle
telomeres - shelterin
TTAGGG are bound by shelterin-telosome protein complex (protection + regulation of telomeres);
subtelomeric regions are adjacent to telomeres, also repetitive DNA
telomeres - methylation
subtelomeric DNA is VERY methylated
- these modifications at telomeres and subtelomeres negatively regulate telomere length (prevent telomerase activity)
