DNA Replication Flashcards
origins of mutations: human males vs females
human males: much more divisions in germ cells
human females: fewer divisions in germ cells
frequency of mutations: (male sperm 10-50X female egg)
DNA replication
- enzymatic/structural/regulatory
enzymatic: nucleic acids as substrates
- DNA: restriction enzymes, topoisomerases, telomerases, methylases, polymerases, etc
- RNA: tRNA synthetases, polymerases, polyadenylases
structural: change in DNA/RNA structure
- helicases, histones, TBP (tata-binding proteins)
regulatory: binding to nucleic acids
- in both transcription (DNA binding) and translation (RNA binding)
Type of protein-nucleic acid interactions (4)
- ionic
- H bonds
- van der waals
- hydrophobic interactions
*also stabilize tertiary and quaternary protein structures
replication is _____ (style of conservation)
semiconservative
replication origin (prok vs euk)
prok: bacterial genomes and plasmids often have only 1
euk: multiple origins
replicon meaning
entire region of DNA replicated from one origin
(a piece of DNA which replicates as a single unit)
experiment that confirmed bidirectionality in DNA replication
pulse-chase experiment
- pulse of ^3H-T (tritium isotope), followed by “chase” of unlabeled T
- light up areas showed bidirectional growth (going in two directions)
discovery of enzymes in DNA rep
- studied prok w genetic and biochemical approaches
- make a mutant pop, screen for DNA rep mutants
- purify enzymes needed for rep
- assay: add labelled dNTP to necessary DNA rep enzymes, template DNA, and measure how much label is in new DNA
what does DnaA do in DNA rep?
initiation
what do single-strand binding proteins do in DNA rep?
they sheathe strands (so don’t reanneal)
what is helicase AKA?
DnaB
what is primase AKA?
DnaG, RNA polymerase
what are clamp loader and sliding clamp AKA?
clamp loader - DnaC
sliding clamp - beta clamp
replisome meaning
all the proteins that function at the replication fork, as part of DNA synthesis
3 problems in copying DNA by DNA pol:
- DNA pol can’t break inter-chain H-bonds at point of origin (need other molecules)
- DNA pol can’t start chains, only elongate (needs primer @3’OH)
- DNA pol can only add from 3’OH end (chain always grows in 5’-3’ direction)
3 ways to generate 3’ ends (for DNA rep), used by diff organisms
specific RNA pol
- synthesize small segment of RNA
- in DNA rep for prok and euk
nicked DNA
- happens in rolling circle replication; some phages do this
primed nucleotide
- some viruses can do this; also at end of euk rep (telomerase)
leading strand vs lagging strand
leading strand - continuously made from a single primer, 5’-3’
lagging strand - DIScontinuous, from multiple primers, also 5’-3’
what are okazaki fragments, what are they joined by?
lagging strand segments
joined by DNA pol I and ligase
how many replication forks in replication bubble?
2 replication forks in 1 replication bubble
processivity meaning
how fast and continuous the polymerase copies (high processive pol are more efficient)
initiation of replication in E. coli (origin of rep name, bp#, characteristic)
OriC, ~245bp
A-T rich 13 bp (3)
repetitive 9 bp (DnaA boxes, 4)
initiation depends on ______ of oriC
methylation
initiation depends on methylation of oriC:
- N6 of adenine is methylated in sequence GATC (copies exist in oriC)
- note: methylations can happen on C and A residues in DNA (prok)
methylation is epigenetic/trans-generational?
epigenetic
- template keeps methyl but new strands don’t, so not passed to future strands (hemi-methylated DNA)
what methylates origins for initiation
Dam methylase
DnaA function, full desc in E. coli DNA rep
- initiates replication at oriC
- recognize 4 DnaA boxes (9bp) in oriC
- 10-20 DnaA proteins and 4 DnaA boxes form an initial complex
- starts replication only if DNA is NEGatively supercoiled (easy to unwind)
– opens at the 13bp, NEEDS ATP
DnaB function, full desc in E. coli DNA rep
what protein escorts it?
AKA helicases!
- separate strands, NEED ATP
- hexamer, can clamp around either ss of DNA
- NEEDS DnaC to escort DnaB to DnaA (form pre-priming complex or pre-primosomal complex)
- moves toward ds region of rep fork
- processive (doesn’t fall off until end of strand or unloaded)
what does SSB stand for?
single-stranded DNA-binding protein
what does SSB (single-stranded DNA binding protein) do?
- small protein that binds to ssDNA
- cooperative binding: binding of one molecule promotes binding of next one
- prevents strands reannealing and internal pairing (no hairpins)
DnaG AKA, function, full desc in E. coli DNA rep
AKA DNA primase
- makes RNA primers for DNA pol to build off
- lagging strand primase (DnaG) works with DnaB (helicase) to form next priming site (PRIMOSOME)
what happens at the end of initiation?
binding of DNAPIII, polymerization starts
are DNA pol I major enzymes for DNA rep in vivo? why or why not?
no, some mutants result in viable and normal rate DNA rep
what is the most important polymerase in DNA rep?
DNA pol III - key enzyme, synthesis of DNA from RNA primers
what does DNA pol II do?
repair
how are nucleotides added? (movement of a molecule)
addition of nucleotide triphosphate;
alpha phosphate connects backbone while base pairs match. other 2 phosphates disconnect (catalysis)
how many daltons in pol I and pol III?
pol I: 1 polypeptide, 109kDa
pol III: 10 diff polypeptides, >600kDa
what is DNAPIII’s core made of? (3 subunits & their functions)
alpha - active site for base addition (5’-3’)
epsilon - 3’-5’ exonuclease (activity that proofreads)
theta - function unclear
what is function of beta subunit in DNAPIII
beta subunit - functions as a clamp; associate with core at 3’ end of growing strand, increases processivity of polymerase (250-1000nt/sec)
what is function of gamma complex in DNAPIII, parts, what proteins does it interact w/
- multiple subunits
- shared by two core polymerases
- loads and unloads beta (clamp) onto DNA template (NEEDS ATP)
- includes tau (2) to connect 2 pol III core (dimerization) -> leading/lagging are coordinated
what direction is DNAPIII’s proofreading in?
3’-5’ exonucleolytic proofreading, high fidelity
