Lecture 15 Flashcards
(31 cards)
Topoisomerase are essential for DNA replication
under or overwinding makes strained DNA
opening it up makes it strained
W number will go up
becomes positive, push all positive supercoils in front of replication fork
positive supercoils have opposite effect of negative supercoils
DNA topoisomerases relax positive supercoiling in front of the fork
Topoisomerase 1A can only relax negative supercoiling- what’s the point?
other topoisomerases do positive- important for separating chromosomes, etc.
DNA polymerization requires an initiating primer
DNA dep dna pol cannot extend a DNA chain from nothing- they can only add nucleotides to a free OH
RNA polymerases don’t need 3’ hydroxyl, can just start synthesizing dana replication
special rna polymerase called primase, used for initiation of dna synthesis. synthesize rna; have dna pol come in and synteshize off of that
DNA polymerase I has open and closed conformations
Like a hand: fingers and thumb apart: open. Fingers and thumb together: closed
dna pol one was the first one found
palm is the active site for polymerization
dna comes in, closes over (induced fit) correct base comes in, better fit than if yo put in the wrong one. Helps select correct nucleotides
Mg 2+ ions importance
Mg helps away leaving group. Good because specifically binds phosphate and has two charges, better counter ion than just one charge
Dideoxy (dd)NTPs
Synthetic nucleotides that lack 3’ OH
(dna)n residues + ddNTP -> (dna)n +1 residues + PPi (lacks 3’ OH)
Chain terminators0 no hydroxyl to attack next. Useful for dna sequencing
Chain terminated because no additional nucleotides can be added to the 3’ position!
ddNTPs are NOT normally found in nature, but they are essential tool for DNA seq and many other analytical methods.
ddCTP chain termination in in the active site of DNA pol 1
Result: can’t do attack, so it stops and falls off
ddNTPs in DNA sequencing: sanger method
Technique called sanger sequencing
use ddNTP, chain is terminated and marked with a 32 P
(ex: use 2,3- dideoxycytidine-5’alpha-(32P)- triphosphate
Use gel, see different lengths in fragments that end in C, T, etc.
Can read sequence of dna in order using gel
Can also do sanger sequencing with fluorescent dye terminators
Products of mixed sequencing reaction containing 4ddNTPs
advance in technology
Acyclovir (ACV), an important antiviral (pro)drug
ACV is a key drug used against herpes viruses (especially), cytomegalovirus etc
in infected cells, ACV is activated by ddephosphorylation making it a dGTP mimic
ACV-TP, the active form, lacks a 3’ OH, so it’s a chain terminator
ACV is very effective and has low toxicity because it is a much better substrate for viral TK (1) and viral dna pol (4) than for human TK or polymerases
effective on viral rna but not ours
lacks sugar and phosphate
Triphosphate form activated, can get added, doe snot have sugar. It’s a chain terminator, not a substrate for our enzyme so it’s great.
Reaction drawn in notes
DNA polymerization requires an initiating primer which can be made from either DNA or RNA
dna dependent dna polymerases cannot extend a dna chain from nothing- they can only add nucleotides to a free OH
rna polymerases do not require an initiating primer
Representative enzymes needing primer: dna pol making dna, rna pol making rna out of dna
not requiring: rna pol using dna, rna pol using rna
What chemical group would you expect to find at the 5’ terminal position of a typical RNA primer and why?
FIND OUT
The lagging strand is synthesized as a series of short okazaki fragments
Each okazaki fragment requires a new primer. Primate is the complex of enzymes that make these. Okazaki pulse chase experiment. Add radioactive thymidine run out on a gel
Later times, okazaki fragments would be joined together
Same time you make leading strand, you are synthesizing short pieces on lagging strand. Must have a way of connecting these together.
Division of labor among prokaryotic DNA polymerases
Dna pol 1, 2, 3 in prokaryotes
eukaryotes: have 13 or fourteen diff polymerases even leading and lagging strands have different polymerases
all reactions and mechanisms similar regardless of pork or euk or which pol
DNA pol 1 in prok
erases primer and fills in gaps on lagging strand (okazaki fragments)
dna pol 1 does dna synthesis.. a mutant of dna pol one could not synth dna at all, but e coli with that mutation were perfectly fine. means e coli must have someone else who can do the job. not the main guy
Dna 2 (error prone polymerase)
dna repair
dna pol 3
primary enzyme of dna synthesis
the main guy in dna synth
DNA replication: lagging strand synthesized as a series of short okazaki fragments
A: RNA primers are laid down by a special DNA dependent rna pol called primase
B: okazaki fragments are elongated by dna pol III
C: gaps between the okazaki fragments are sealed by dna pol I and dna ligase
Nucleases:
phosphoesterases that cleave dna and rna
substrates: DNAses, RNAses
General reaction: phosphoester + H2O -> acid (R-Pi) + alcohol (R-OH)
-M2+ cofactor required, usually magnesium
-some nucleases leave 3’ Pi while others leave 5’ Pi
Exonucleases
Chew away on free ends
two main types, chew in opposite directions
- 5 to 3 enzymes require free 5’ end on substrate
-3 to 5 enzymes require free 3’ end
Endonucleases cut in the middle of a strand or duplex
- some are relatively non-specific (will cleave any sequence)
- site specific nucleases cleave only at specific sequences
- some cut a single strand; others cut both strands
Function of DNA pol 1 in gap repair
makes DNA to chew it up- getting rid of little rna primers
have different okazaki fragments, have space between them, have rna primer. dna pol chews away and synth at the same time to fill in the gaps.
In addition for its polymerase activity, dna pol 1 has 5 to 3 exonuc activity that removes RNA and dna primers
very end, have a small nick which dna ligase fixes
DNA ligase seals the nick
1)ligase active site lysine forms covalent activated intermediate with NAD or AMP
2) adenosine 5’ diphosphate attached to 5’ end of nick
note 5’5’ pyrophosphate linkage
3) nucleophilic attack by 3’ OH seals nick, regenerates enzyme and AMP
The pol III holoenzyme is an asymmetric dimer that catalyzes both leading and lagging strand synthesis
SSB - single stranded dna binding protein. SSB keeps melted DNA from re-annealing and protects the exposed bases
Note the looping of the lagging strand DNA
to synthesize the lagging strand, must pull out so you can have something to synthesize on
Especially an issue on lagging strand: coat ss parts with SS binding protein, enables you once you have separated the strand to have it not come back together with template
Sliding clamp is a processivity factor
It keeps the polymerase complex from falling off the DNA
Sliding clam, dimer of two different halves that forms doughnut around dna. Many basic/positively charged residues
puts DNA on tightly, but not so tight that it cannot move; it can slide
To get it on: must be able to open it and put it on. Issue on the lagging strand because you are constantly having to open and close and jump
1 sec- synth 1000 nuc, length of one okazaki
Regulation fo DNA replication
3 questions
1) not how it occurs, but where it occurs? In ac ell, could be a compartment, euk will be nucleus, could mean where like which tissue or cell type
2) when? cell cycles, times dividing and not dividing. Only want to synthesize dna when dividing. only want to replicate the chromosome once per division
3) how much? for dna, just once per cell cycle. for transcription or translation can regulate to make a little or a lot of something
