Lecture 15

Question 1

Topoisomerase are essential for DNA replication

Answer 1

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.

Question 2

DNA polymerization requires an initiating primer

Answer 2

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

Question 3

DNA polymerase I has open and closed conformations

Answer 3

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

Question 4

Mg 2+ ions importance

Answer 4

Mg helps away leaving group. Good because specifically binds phosphate and has two charges, better counter ion than just one charge

Question 5

Dideoxy (dd)NTPs

Answer 5

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

Question 6

ddNTPs in DNA sequencing: sanger method

Answer 6

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

Question 7

Acyclovir (ACV), an important antiviral (pro)drug

Answer 7

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

Question 8

DNA polymerization requires an initiating primer which can be made from either DNA or RNA

Answer 8

dna dependent dna polymerases cannot extend a dna chain from nothing- they can only add nucleotides to a free OH

Question 9

rna polymerases do not require an initiating primer

Answer 9

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

Question 10

What chemical group would you expect to find at the 5’ terminal position of a typical RNA primer and why?

Answer 10

FIND OUT

Question 11

The lagging strand is synthesized as a series of short okazaki fragments

Answer 11

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.

Question 12

Division of labor among prokaryotic DNA polymerases

Answer 12

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

Question 13

DNA pol 1 in prok

Answer 13

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

Question 14

Dna 2 (error prone polymerase)

Answer 14

dna repair

Question 15

dna pol 3

Answer 15

primary enzyme of dna synthesis

the main guy in dna synth

Question 16

DNA replication: lagging strand synthesized as a series of short okazaki fragments

Answer 16

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

Question 17

Nucleases:

Answer 17

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

Question 18

Exonucleases

Answer 18

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

Question 19

Endonucleases cut in the middle of a strand or duplex

Answer 19

• 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

Question 20

Function of DNA pol 1 in gap repair

Answer 20

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

Question 21

DNA ligase seals the nick

Answer 21

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

Question 22

The pol III holoenzyme is an asymmetric dimer that catalyzes both leading and lagging strand synthesis

Answer 22

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

Question 23

Sliding clamp is a processivity factor

Answer 23

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

Question 24

Regulation fo DNA replication

Answer 24

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

Question 25

In addition to being semiconservative, and discontinuous, DNA replication is bidirectional- two forks diverge from each origin

Answer 25

Most about how you initiate or start the process
replication of a circular DNA duplex in a bacterium will make “replication bubbles”
Bidirectional synth of dna: two replication forks
open at origin, put two diff replication fork machineries, each going in different directions
origins are at rich- less stacking energy, easier to pull apart
origins typically have mainly a’s and t’s

Question 26

Where? - initiation of dna replication

Answer 26

Initiation of DNA replication
The circular e coli chromosome initiates replication at a single location
The dna sequence is called ori C
Ori C is specifically bound by a set of proteins that melt the DNA and place two replication factories on the dna, facing in opposite directions

Question 27

When, how often?

Answer 27

bacterial chromosomes can initiate replication more than once in a cell cycle
under these conditions, each daughter cell already contains a chromosome that has been partially replicated
e coli divides every 20 min- every 20 min it will synthesize its own chromosome
20 min, only will have gone halfway through chromosome
get layering, so to speak

Question 28

why do bacteria do this?

Answer 28

FIND OUT

Question 29

How does it end?

Answer 29

Two new chromosomes are connected- how do you separate them? Topoisomerase type 2
(replication without unwinding, get catenated chromosomes)

Question 30

Multicellular eukaryotes face another problem. They usually have a lot more dna than bacteria.

Answer 30

Polymerase slower as well. 1 month to synthesize chromosome in human cells. Things like red blood cells need to be faster than a month. answer: have many origins of replication

Question 31

Another when problem: if you have hundreds or thousands of origins, how do you initiate at each one only once per cell division cycle?

Answer 31

Ways to turn on each one once…. telomerase? check and see if this slide was finished in class