Lecture 18

Front 1

Meselson-Stahl Experiment

Back 1

Replication is proved to follow a semiconservative model and produces a first generation-daughter hybrid molecule.

This is proven by the presence of a hybrid molecule with one heavy and one light chain.

Front 2

Requirements of DNA polymerases

Back 2

1) Temple strand to copy
2) Primer strand with 3' OH group
3) dNTP

Front 3

Function of DNA polymerase

Back 3

1) Nucleophilic attack by 3 'OH group
2) Phosphodiester bond formation
3) 5' to 3' synthesis

Front 4

Pyrophosphate

Back 4

Release after the phosphodiester bond formation and lends further favorable energy to the reaction.

Front 5

How is accuracy or fidelity ensured in replication?

Back 5

Geometry: the active site only accommodates the correct base paring

Front 6

Polymerization Rate

Back 6

Nucleotides added per second

Front 7

Processivity

Back 7

Nucelotides added before polymerase dissociates

Front 8

Which Polymerase replicates the genome? Why?

Back 8

Pol III because high polymerization rate and processivity.

Front 9

Proofreading

Back 9

Occurs in the opposite direction to synthesis: 3' to 5'.

Can be carried by all three of the polymerases.

Improves accuracy 100 to 1000 fold from errors occurring 10^(4-5) to 10^(6-8)

Front 10

Major stages of DNA replication

Back 10

1) Initiation
2) Elongation
3) Termination

Front 11

Origin

Back 11

A specific point or points on a chromosome where replication begins and then moves bidirectionally.

In bacteria, there is a single origin of replication.

High conserves with several key region:
1) R and I sequences bind to DNA A.
2) DUE

Front 12

DUE

Back 12

DNA Unwinding Element

AT rich region which is important for 1) loading the replication helicase (DNA B) 2) protein that function to bend the DNA

Front 13

DNA A

Back 13

Bond to ATP

Binds to R and I sites at the origin.

Forms a helix forcing the DNA into a supercoil.

Supercoil facilitates unwinding resulting in an opening at DUE sequence.

Front 14

DNA B

Back 14

Helicase that unwinds the DNA.

Hexamer ring.

Conformation change causes open form and closes around DNA.

Loading facilitated via DNA C - ATP

Front 15

SSB

Back 15

Single Stranded Binding protein which stabilizes the single strand

Front 16

Primase

Back 16

Set up RNA primer providing the primer strand with 3' OH required by DNA polymerase.

Front 17

How are two okazaki fragments fused together?

Back 17

1) Removal of primer (5' to 3' exo activity) and filling of DNA (5' to 3' polymerase activity) by Pol I.
2) Sealing nick by ligase.

Front 18

Ligase function/mechanism

Back 18

Connect okazaki fragments

1) Adenylation of enzyme.
2) Activation of 5' phosphate
3) Nucleophilic attack by the 3' OH

Front 19

DNA gyrase

Back 19

Topoisomerase II that relieves strain from unwinding.

Occurs at the elongation step of replication.

Front 20

Clam loading complex

Back 20

Holds together two core DNA Polymerase molecules and DNA B helicase.

Loads on a Beta clamp, forcing it in an open conformation.

Coordinates the movement of the two strands by looping the lagging strand.

Front 21

Beta clam

Back 21

Ensure the processivity, therefore without it the polymerase would fall off much more quickly.

Dimer of two subunits.

Front 22

Ter Sequences

Back 22

Form traps for the replication forks as the move along.

This is down by the interaction of the Tus protein with DNA B helicase.

Front 23

Replication rate in prokaryotes vrs. eukaryotes

Back 23

1000 fold slower in eukaryotes.

10 fold slower rate and 100 fold greater length.

Front 24

Eukaryote structure analogous to DNA B helicase

Back 24

MCM2-7

Front 25

What complexes in the Pre-RC must dissociate for replication to begin in eukaryotes?

Back 25

Cyclic dependent kinases dephosphorylate CD6 resulting in the dissociation of

CD6 and CDT1 protein

Front 26

What's important to note about multiple origins of replication?

Back 26

Activation is signal dependent

--so that all DNA can be replicated at the same time in the S phase.

Front 27

Telomeres

Back 27

Repeated sequences that stabilize the ends of chromosomes.

Normally, TG sequences.

Front 28

Telomerase

Back 28

Adds telomeres to chromosome ends.

RNA component of the enzyme provides the template for the telomeres.

RNA dependent DNA polymerase.

Front 29

Mutation: Silent

Back 29

No effect on gene function.

Front 30

Mutation: Deleterious

Back 30

Impairs gene function

Front 31

Mutation: Advantageous

Back 31

Enhances gene function

Front 32

Three different kinds of mutations

Back 32

Base Insertion
Base Deletion
Base Subsitution

Front 33

Mutations: Base Substitutions: Transitions

Back 33

Pur to Pur
Pyr to Pyr

Front 34

Mutations: Base Substitutions: Transversions

Back 34

Pur to Pyr
Pyr to Pur

Front 35

Examples of deaminating agent

Back 35

Nitrous acid precursos:
1) Sodium Nitrate
2) Sodium Nitrite
3) Nitrosamine

Front 36

Deamination

Back 36

Loosing an amine group.

For example, cytosine loosing its amine group to form uracil.

Front 37

Why is it a good reason to have thymine instead of uracil?

Back 37

Deamination of thymine would form uracil, therefore the mutation would be unrecognizable.

Front 38

What does UV radiation cause?

Back 38

The formation of cyclobutane thymine dimer, a kind and the stopping of replication.

Front 39

Depurination

Back 39

The base is hydrolyzed off of the ribose via alkylating agents.

Front 40

Alkylating agents

Back 40

Methylates guanine to methylguanine which no longer can bind to cytosine and binds to thymine.

After replication... this then introduces a new AT mutation.

Front 41

Dam methylase

Back 41

Methylates daughter strands after replication.

Front 42

What is the time period for mismatch repair?

Back 42

After replication and formation of the daughter strand but before methylation of the daughter strand by the Dam methylase at the N6 adenine.

Front 43

MutL-MutS complex

Back 43

Finds mismatch and binds
DNA is threaded throw till methylated adenine is found (therefore identifying the correct strand).

Front 44

MutH

Back 44

Cleaves the unmodified strand

Front 45

In the mismatch repair mechanism, what polymerase is used? What strand does it copy?

Back 45

Pol III copies the methylated strand