Dna Replication (Lecture 3)

Front 1

3 major pathways of information flow in the cell

Back 1

1. replication: info passing from one DNA to another DNA (making new daughter strand)

2. transcription: info passing from DNA to RNA

3. translation: info passing from RNA to proteins

Front 2

DNA replication

Back 2

-copying one DNA molecule to another DNA molecule

-part of cellular division

-ability to correct mistakes

-has to be fast (longer it takes, more opportunity for something outside to come in and affect)

Front 3

semiconservative

Back 3

-results in exact duplicates of the original dsDNA molecule

-each strand is a template for synthesis of new strand

-H bonds break and two DNA strands separate

Front 4

semiconservate vs conservative vs dispersive replication

Back 4

1. single strand conserved

2. both strands conserved

3. chopped up and each gets bits

Front 5

Messelson-Stahl experiment

Back 5

-proved DNA is replicated semiconservatively

-DNA in E. coli labeled with 15N (15N/15N)

-bacteria transferred to 14N medium; one round of replication (15N/14N)

-second round of replication (14N/14N + 14N/15N)

-subsequent replications (14N/14N + 14N/15N but much more 14N/14N present)

Front 6

one molecule of DNA is one or two strands?

Back 6

two

Front 7

origin of replication

Back 7

-where DNA synthesis begins

-prokaryotes: oriC

-eukaryotes: ORC (origin recognition complex)

-bidirectional synthesis: creates a replication bubble in which synthesis occurs in both directions

Front 8

DNA polymerases

Back 8

-large enzymes that mediate DNA replication

-primer dependent: can't start replication without short DNA or RNA molecules with a free 3'-OH

-add nucleotides one a a time to the 3' end of a primer

-matches new nucleotides based on a template strand (A/T, G/C)

-always goes 5'-3' direction

Front 9

replication fork

Back 9

-DNA synthesis has to take place simultaneously but in opposite directions on the two template strands

-lagging has to do 5'-3' but that's opposite of the direction the bubble is opening up

-pic in notes shows primers and oriC

Front 10

leading strand

Back 10

-proceeds in same direction as the movement of the replication fork

-synthesized continuously

-requires only one RNA primer

Front 11

lagging strand

Back 11

-proceeds in opposite direction of the movement of the replication fork

-laid down in intervals as the replication fork moves

-DNA synthesized in short Okazaki fragments

-requires multiple RNA primers

-RNA primers must be replaced by DNA (DNA polymerase I) and the gaps repaired (DNA ligase)

Front 12

bacterial DNA replication

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1) theta replication

2) rolling-circle replication

Front 13

shape of bacterial chromosome

Back 13

circular

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theta replication

Back 14

--> 2 identical circular DNA molecules

Front 15

rolling-circle replication

Back 15

-typically how plasmids replicate in cell division

-outer strand is nicked by an enzyme and free 3'OH end gets extended by polymerase
-outer strand peels off as new daughter strand is produced
-second strand circularizes and is replicated

--> multiple circular DNA molecules

https://www.youtube.com/watch?v=fB1OkshHAVw

Front 16

plasmids

Back 16

-small circular dsDNA found in bacteria

Front 17

bacterial replication mechanisms

Back 17

1. initiation

2. unwinding

3. elongation

4. termination

Front 18

bacterial replication initiation

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-step one

-single replication origin (oriC)

-initiator proteins (DnaA) bind to oriC and cause a short segment of DNA to unwind

-leads to initial bubble, opening up dsDNA molecule at the origin

Front 19

bacterial replication unwinding

Back 19

-step two

-helicase (DnaB): binds to the short stretch of ssDNA - binds to the lagging strand template and moves 5'-3 breaking H bonds to move the replication fork

-topoisomerase: enzyme that manages DNA supercoiling, reducing tension at the head of the bubble, works ahead of the bubble

-single-strand-binding proteins (SSBs): attach and stabilize the single-stranded DNA during replication, protect and keep separated, prevent from reannealing

-primers: elongation requires a 3'OH group, primase synthesizes a short RNA primer and places it at the origin - leading strand = 1 primer, lagging strand = primer before every Okazaki fragment

**VIDEOS**

Front 20

DNA helicases

Back 20

-enzymes that break H bonds between two strands of DNA

Front 21

bacterial replication elongation

Back 21

-DNA polymerase: elongates the polynucleotide strand (five types)

1. pol I: removes RNA primers and fills in the gap

2. pol III: major replication enzyme (most important)

3. pol II, IV & V repair DNA damage (distortions in new molecules , incorrect nucleotides, etc)

-DNA ligase: seals the nick in the sugar-phosphate backbone between the 3'OH of the last nucleotide added by pol I and the 5'P group of the first nucleotide added by pol III - catalyzes a phosphodiester bond in gap between pol I and pol III's work

Front 22

DNA polymerase I

Back 22

-replaces all RNA primers used to initiate elongation

-nick remains in the sugar-phosphate backbone of the new DNA strand between the 3'OH of the last nucleotide added by pol I and the 5'P group of the first nucleotide added by pol III

Front 23

DMA polymerase III

Back 23

-enzyme that catalyzes the synthesis of DNA by using a template strand

-synthesis occurs 5'P-3'OH on daughter strand (so 3'-5' on parent)

-proceeds along a single-stranded molecule of DNA

-free nucleotides attached to complement the existing base (A-T or G-C) using H bonds

-new nucleotide is attached to the previous nucleotide on the daughter strand with a phosphodiester bond

Front 24

nuclease activity

Back 24

-DNA polymerases also have nuclease activity: ability to break phosphodiester bonds in sugar-phosphate backbone

-built-in mechanism for correcting rare errors in DNA synthesis

-if pol III adds an incorrect nucleotide it can cut out wrong nucleotide and insert correct one

-two types of nucleases: exonucleases (remove nucleotide from end of chain) and endonucleases (break bonds within a chain)

-ALL have exonuclease activity in 3'-5' direction (just backspace)

-reduces frequency of mutation

-replication is very accurate: <1/billion nucleotides

Front 25

bacterial replication termination

Back 25

-can occur when two replication forks meet

-in some cases specific termination sequences block further replication (e.g. Ter sequence: Tus protein binds to Ter sequence which blocks the movement of the helicase, stalling the replication fork and preventing further DNA replication

-not a lot known about mechanism

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key differences between eukaryotic and prokaryotic replication

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1. multiple origins of replication (a lot more DNA)

2. slower rate of DNA synthesis (still rapid though)

3. difference in nomenclature of proteins used

4. telomere replication because linear DNA

Front 27

eukaryotic DNA replication

Back 27

-eukaryotic chromosomes are linear and much larger

-require a different mechanism from prokaryotic to replicate their DNA

-DNA is copied quickly with use of thousands of origins of replication creating thousands of replication bubbles

Front 28

eukaryotic replication mechanism

Back 28

-occurs in a fixed location: enzymes (helicase and polymerase) are stationary and DNA moves through

-polymerase doesn't move along DNA strand

-template is threaded through DNA polymerase with newly synthesized strands emerging

-lagging strand loops out and comes around

-little SSB protein balls keeps strands separated so don't H bond back together

https://www.youtube.com/watch?v=-mtLXpgjHL0

*good illustration in book

Front 29

eukaryotic replication initiation

Back 29

-at each origin, a multiprotein origin of recognition complex (ORC) binds to initiate unwinding - timing is regulated so that each segment of DNA (and each gene) is replicated only once per cell cycle - lots of bubbles

-polymerases interact with the proliferating cell nuclear antigen (PCNA) - functions as a sliding clamp to help bind the polymerases to the DNA strand and maintain contact - ensuring rapid/efficient polymerization

Front 30

eukaryotic replication unwinding & elongation

Back 30

-similar to bacterial replication mechanism but different proteins

Front 31

eukaryotic DNA polymerases

Back 31

-different from bacterial

-large number involved in replication, recombination, and DNA repair

-pol alpha: lays down primers, initiates nuclear DNA synthesis and DNA repair

-pol delta: lagging strand synthesis, DNA repair

-pol epsilon: leading strand synthesis

Front 32

eukaryotic replication termination

Back 32

-critical difference with prokaryotes and eukaryotes

-problems with completing replication - gap left by removal of the last RNA primer that DNA polymerase can't fill in

-if chromosomes became shorter, over time would lead to cell death

--> telomeres

Front 33

telomerase mechanism

Back 33

-telomeres: copies of short repeated sequences - not functional genes, just a buffer/protective region

-tetrahymena (protozoan): telomeric repeat = 5'-TTGGGG-3'

-telomerase enzyme: lengthens chromosome ends by extending the template strand telomere 5'-3' so the pol alpha can add an RNA primer to that end so the important parts of the chromosome get copied

-telomerase for tetrahymena = AACCCC repeat

https://www.youtube.com/watch?v=y_pkaKDHpWs

https://www.youtube.com/watch?v=AJNoTmWsE0s

Front 34

telomerase enzyme

Back 34

-present in germ cells, early embryonic cells, certain proliferative somatic cells (bone marrow, intestinal) - cells that become differentiated

-most undergo continuous cell division

-most somatic cells exhibit little/no telomerase activity, so chromosomes become progressively shorter, limited number of divisions

-defective telomerases lead to premature aging

-anti-aging therapy?

-Dolly: her genetic material came from a six-year-old sheep, so her telomeres were slightly shorter and she died young