Dna Replication, Repair And Recombination

Front 1

What's the difference btwn A-DANA and B-DNA?

Back 1

A is shorter and wider (dehydrated) and has a C-3' sugar pucker

B is longer and narrower with a C-2' sugar pucker (generally cellular DNA is Bform)

Front 2

What causes the major and minor grooves?

What is the significance of the H-bonding the grooves can form?

Back 2

base pairs are not diametrically opposite eachother. Major grove has the bigger side of the base pair (Thy-Methyl) and minor groove has the smaller side (pyrimidine O-2 and purine N-3)

Significance of grooves' side and H bond potential is how proteins can interact with specific DNA sequences.

Front 3

What is a good H bonder in the minor groove?

What is a good H bonder in the major groove?

Back 3

N-3 of G or A (minor groove)

N-7 of G and A (major groove)

Front 4

Is DNA perfectly uniform?

Back 4

No way. There are ranges (28-42 degrees vs. the 36 degrees of watson crick model) of degrees of coil and they have local deviations

There can be:
propeller twisting
Z-DNA
Supercoiling

Front 5

What is propeller twisting?

Back 5

base pairs are not perfectly coplaner and the amount of this depends on the base pair sequence.

Front 6

What is Z DNA?

Back 6

left handed instead of right handed double helix.

occur in short sequences of oligonucleotides that alternate (dCGCGCGCG)

Need high salt concentration to alleviate static repulsions, but these guys are present in cells so they must be there to perform a specific function where it doesn't have to be so super salty.

Front 7

Describe the shape of A, B and Z DNA.

Back 7

A=broadest

B=Intermediate

Z=Narrowest

Front 8

Describe the screw sense of A, B and Z DNA.

Back 8

A= right handed

B= right handed

Z= left handed

Front 9

what are the grooves like of A DNA?

Back 9

major= narrow and deep

minor= broad and shallow

Front 10

What are the grooves of B DNA?

Back 10

major= wide and deep

minor= narrow and deep

Type B's are both deep

Front 11

What are Z DNA grooves like?

Back 11

major=flat

minot = narrow and deep

Front 12

What happens if you unwind B-DNA a couple of turns?

Back 12

when it ligates it can have 2 forms

23 turns and an unwound loop

or

25 turns and a negative superhelix.

Front 13

What does supercoiling accomplish?

Back 13

much more compact DNA and it moves faster in electrophoresis.

Front 14

What is the linking number?

Back 14

# of times a strand of DNA winds in he right-handed direction when the helix is constrained to lie in a plane.

Front 15

What is a topological isomer (topoisomer)?

Back 15

molecules differing only the LK

Front 16

How can topoisomers be interconverted?

Back 16

only by cutting one or both strands and rejoining them

This is the role of topoisomerases.

Front 17

What are DNA with the same LK but that are geometrically different?

Back 17

have differing Tw and Wr numbers.

Front 18

what is twist?

What is writhe?

Back 18

Tw = helical winding of DNA strands around eachother

Wr= measure of coiling of axis of double helix = supercoiling

Front 19

what is the optimum energy in the expression for the equation that depicts supercoiling?

Back 19

70% of LK expressed in Wr

30% of LK expressed in Tw

Front 20

Does negative supercoiling indicate getting tighter or looser for topoisomerase activity?

Back 20

Means it's getting looser. Most naturally occuring DNA is negatively supercoiled priming it for strand separation for things like transcription and repair.

Front 21

What do Type I isomerases do?

Back 21

catalyze relaxation of supercoiled DNA

thermodynamically favored so no ATP necessary.

alter the LK, cleaves ONE DNA strand, does its thing (replicate, transcribe or recombine) and reseal

Front 22

What do Type II isomerases do?

Back 22

use ATP to create negative supercoils to DNA to make them more compact.

alter the LK, cleaves both DNA strands, does its thing (replicate, transcribe or recombine) and reseal

Front 23

In the cavity of human Type I topoisomerase what is the crucial residue?

Describe how this enzyme works.

Back 23

Tyr 723

DNA is bound to the enzyme cavity.

Tyr 723 OH is NU that attacks a phosphate group on DNA making a phosphoester bond and cleaving the DNA

Now there is a free 5' end

Front 24

Describe what is happening in this image.

Back 24

DNA binds to Type I topoisomerase and Try 723 OH acts as a Nu to attack a Pi group on DNA and forming a phosphoester bond and thus cleaving the strand to leave a free 5' end of DNA

Front 25

What happens once there is a free 5' end of DNA once Type I topoisomerase has cleaved it?

Back 25

Because the freeing of DNA is thermodynamically favorable, the 5' end unwinds some. Then the 5' OH attacks the Try 723 that is bonded to the 3' end of the DNA to reseal the strand.

DNA dissociates from the enzyme.

Front 26

Describe Type II topoisomerase.

Back 26

dimer with 2 internal cavities.

Big cavity has 2 gates (binds G strand tightly and the other gate binds the transported or T strand loosely)

both gates have Tyr residues that mimic type I activity.

Front 27

how does one cycle of the activity of Type II topoisomerase effect LK and WR?

Back 27

one cycle decreases the LK by 2

Front 28

What does novobiocin block?

Back 28

blocks the binding of ATP to bacterial DNA gyrase (the bacterial version of Type II topoisomerase)

Front 29

What does nalidixic acid and cipro block?

Back 29

block the cleavage and resealing of DNA in bacterial gyrase (bacterial version of type II topoisomerase)

UTI's and other infections (anthrax)

Front 30

What does camptothecin block?

Back 30

human anti-tumor agent

blocks human topo I by stablizing the enzyme when it's Tyr is bound to the DNA and so the strand unwinds way more and endlessly because there is no releasing and resealing.

Front 31

What does a "template-directed enzyme" mean?

Back 31

Each nucleotide coming in first forms a connection with the template base pair and then it is connected to the nucleotide next to it.

Front 32

Which direction are nucleotides added onto the template?

What is the mechanism?

What is needed at the outset?

Back 32

added to the 3' end.

the 3' OH of the terminus gets activated by the DNA polymerase to be the Nu that attacks the a-phos group of the nucleoside triphos that is getting added.

a primer must be in place with a free 3' OH.

Front 33

What is the Klenow fragment?

Back 33

a part of DNA polymerase I in E. coli

Has 3 domains:
1). "fingers and thumb" wrap around DNA helix

2). "palm" is polymerase activity

3). additional 3'-->5' exonuclease domain (proofreading)

Front 34

How do the 2 metal ions work in DNA polymerase mechanism?

Back 34

One metal ion binds the dNTP and 3' OH of primer (activates the OH to be the Nu)

The other metal ion only interacts with the dNTP

The interactions hold and position the stuff properly. Also balance the negative charge that gets created

Front 35

Explain how these metal ions contribute to the mechanism of DNA polymerase.

Back 35

DNA polymerase mechanism:

Two metal ions (Mg2+)
One metal ion coordinates 3'-hydroxyl of primer
Other metal ion interacts with dNTP
Phosphate group of nucleoside triphosphate bridges 2 metal ions
Primer hydroxyl attacks phosphate group to form new O-P bond

Front 36

Discuss how H bonding and nucleoside triphos shape contribute to replication specificity.

Back 36

It's not just H bonding or just shape, but a combo of both.

Even if no H bonds can form, an analog to adenosine will still bind thymidine because the shape.

Front 37

Why is shape so important in DNA polymerase activity?

Back 37

minor groove H-bonding of polymerase ensure a properly spaced base pair has formed (H-bond acceptors are in same position for all Watson-Crick base pairs)

Uses this measure to make sure of a proper fit. If the H bonds don't happen the dNTP gets kicked out.

Front 38

What are 3 features of DNA polymerase that ensure fidelity of DNA replication?

Back 38

minor groove H bonding

Shape of dNTP

"finger" conformational change that clamps down on dNTP and makes sure only a properly shaped base will fit

Front 39

What is primase?

H

Back 39

RNA polymerase

makes the primer with the free 3' OH for the initiation of DNA synthesis

Front 40

How does primase work?

Back 40

Primase synthesizes a short (5 nucleotide) RNA strand that is complementary to one of the DNA strands

Front 41

What is the replication fork?

Back 41

site of DNA synthesis

leading strand is synthesized continually in the 5'-->3' direction by DNA polymerases

The other lagging strand has to make Okazaki fragments and add them on to RNA primers made by primase

Front 42

What are Okazaki fragments?

How do they get linked?

Back 42

small fragments of synthesized DNA that get started on the lagging strand by RNA primer and allow for the 5'-->3' synthesis on this strand.

This means the orignal strand running 3'-->5' is growing too, but the growth is in a 5'-->3' way.

DNA ligase links the Okazaki fragments.

Front 43

What is DNA ligase?

Back 43

joins the ends of adjacent Okazaki fragments

creates a phosphodiester bond between 3' OH and one DNA end with the 5' Pi group of another.

This bad boy only works on ssDNA at the end of a dsDNA so it cannot just link up 2 ssDNA or make a circle or anything like that EXCEPT for T4 bacteriophage ligase which can put 2 blunt ends together

Front 44

What are helicases?

Back 44

use ATP hydrolysis to separate DNA strands

Front 45

What is bacterial helicase PcrA?

Back 45

extensively studied helicase which gives us an idea how they work.

4 domains

A1--P loop NTPase fold (ATP binding and hydrolysis)
(bind ssDNA)
A2--

B1--like A1 but no P-loop (bind ss DNA)
(does NOT bind nucleotides)

B2--

When no ATP is around both A1 and B1 are bound to ssDNA

Front 46

explain the domains and how this helicase PcrA is working to unwind the DNA

Back 46

Helicase mechanism
PcrA domains A1 and B1 bind ssDNA
ATP binding causes cleft closure between domains
Domain A1 slides along ssDNA
ATP hydrolysis causes cleft opening
DNA is pulled from domain B1 to A1
Process repeated to unwind dsDNA

Front 47

Which direction does the PcrA enzyme work on the ssDNA it binds?

Back 47

3' -->5' direction

Front 48

What are some characteristics of polymerases with regard to speed and efficiency?

Back 48

high catalytic potency, fidelity and processivity

(processivity is the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate)

Front 49

What is E.coli DNA polymerase III?

Back 49

replicative polymerase

B2 subunit is what gives it that special something.
Keeps the polymerase associtated with the DNA DOUBLE helix

Looks like a star and acts like a sliding DNA clamp

Front 50

What are clamp loaders?

Back 50

other enzymes that use ATP to pull the DNA polymerase III clamps off the DNA double strand for a sec so the DNA can slide through quickly as its being replicated.

Front 51

What is SSB?

Back 51

single-strand binding protein. Keeps the unwinding DNA strand (ripped asunder by helicases) from reattaching to them selves

Front 52

Describe DNA polymerase holoenzyme and how it keeps the 2 sides coordinated while the leading and lagging sides are synthesized.

Back 52

The enzyme has 2 copies of the polymerase core (one works on leading and one works on lagging strands). These are both linked to a central structure which includes the clamp loader complex and the helicase (DnaB)

Front 53

What is the trombone model?

Back 53

lagging strand loops out in same direction as leading strand. DNA polymerase III working on that strand adds ~1,000 nucleotides and then releases it.

Front 54

What does DNA polymerase I do?

Back 54

5'-->3' exonuclease activity that removes the primer for ligase to eventually come in and link the fragments.

Front 55

What is oriC locus?

Back 55

The origin of replication spot for E. coli.

Has 3 identical 13-nucleotide sequences and then 5 AT binding sites that bond with DnaA

Front 56

What is DnaA origin recognition protein?

Back 56

binds to the 5 AT spots on oriC to start this whole party

c-terminus of each of its monomers binds with the 5 spots

Front 57

What is the pre-priming complex?

Back 57

Once a DNA strand gets bound around DnaA other proteins like DnaB helicase and SSB proteins come in to unzip and keep apart the strands. This whole thing is the pre-priming complex.

Front 58

What is DnaG?

Back 58

the guy that comes to the pre-priming complex and constructs the primer.

Front 59

What's the difference between prokaryotic and eukaryotic replication origin?

Back 59

pros--significantly smaller genetic info that is circular, has only one origin site.

euk--huge genetic info that is linear, lots of chromosomes, multiple origin sites.

Front 60

What is a replicon?

Back 60

starting site for a replication unit (one of the 30,000 in humans)

Front 61

If prokaryotes have the specific sequence oriC what ddo eukaryotes have?

Back 61

ORC's

Not specific sequences but rather broadly defined AT sequences where these complexes are assembled

ORC's have 6 different proteins homologous to DnaA

Front 62

What are licensing factors?

Back 62

recruiters to the ORC of other proteins like
--Mcm2-7 helicase which is the unzipper
--replication protein a (SSB)

Front 63

What is the main processive DNA polymerase in Eukaryotes and how does it get started?

Back 63

DNA polymerase d.

first polymerase a comes in and starts replicating but gets switched out with DNA polymerase d.

Front 64

What are the 3 proteins in DNA repair that are in the MMR systems?

Back 64

MutS (detects mismatch)

MutL (recruits endonuclease)

MutH (said endonuclease)

Front 65

What is hereditary nonpolyposis colorectal cancer caused by?

Back 65

defective DNA mismatch repair

There are mutations in the MMR system proteins

70% risk and constitutes 2-5% of all colorectal cancers.