Watson and Crick's model

-predicts that when a double helix replicates, each of the daughter molecules will have:

1 old strand and 1 newly made strand

new double helix modelled on original, but contain all new nucleotides

->original strand ("conservative"), and a whole new strand

Matthew Meselson and Franklin Stahl's experiments

How did they test the conservative model, dispersive model, and semi-conservative replication model hypotheses?

1.) labeled nucleotides of old strands w/ heavy isotope of nitrogen (15N)

2.) allowed these strands to replicate in a medium containing the lighter isotope of nitrogen (14N)

1.) What would have happened if the conservative model was correct?

2.) dispersive model?

3.) semi-conservative model

How did they test these predictions?

1.) original DNA molecule would contain 15N, all the new molecules would contain 14N

2.) each DNA molecule descended from original would contain mix of 15N and 14N

3.) in first replication, one strand of each molecule would contain only 15N and the other only 14N

in second replication, there would be 2 mixed molecules, two with only 14N

tested these by separating the replicated by density in a centrifuge

-any new nucleotides in the new strands would be lighter than the old strands because they'd contain 14N

1. the first replication in the 14N medium -> single band of dNA

= mix of 15N and 14N

NOT CONSERVATIVE

2. second replication produced 2 bands, one containing light DNA (14N) and one containing hybrid DNA

NOT DISPERSIVE

Origin of replication

1. How are they recognized?

2. What happens at these sites. involving "bubbles" and DNA polymerase?

What is the rate of replication?

3. Where is it in bacteria?

1. where replication of DNA molecule begins

may be hundreds or thousands of origin sites per chromosome

2. recognized by replication enzymes which separate the strands, forming a replication "bubble"

3. proceeds in both directions until entire molecule is copied

at each end of a replication "bubble" are replication forks

- the replication bubbles elongate as the DNA is replicated, and eventually fuse

DNA polyermases catalyze the elongation of new DNA at a replication fork

as nucleotides align with complementary bases along the template strand, they are added to the growing end of the new strand by the polymerase

500 nucleotides per second in bacteria, and 50 per second in human cells

3. In bacteria, it is a single, specific sequence of nucleotides that forms the origin.

nucleoside triphosphates

1. What happens to form pyrophosphate?

2. What drives the polymerization of the nucleotide to the new strand?

raw nucleotides with a nitrogen base, a sugar (deoxyribose), and a triphosphate tail

1. as each nucleotide is added to the new strand, the last 2 phosphate groups are hydrolyzed to form pyrophosphate

2. the exergonic hydrolysis of pyrophosphate to 2 inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand

antiparalellism

describes the strands in the double helix: the sugar-phosphate backbones run parallel to each other, but in opposite directions

each DNA strand has a 3' end with a free hydroxyl group attached to the sugar, and a 5' end with a free phosphate group attached to that sugar

- the 5' -> 3' direction of one strand runs counter to the 5' ->3' direction of the other strand

DNA polymerase

Where can it add nucleotides?

What problem does this create?

to the free 3' end of a new DNA strand

can only elongate at the 3' end

problem at replication fork because 1 parental strand is oriented 3'->5' into the work, and the other is 5' ->3' into the fork

DNA polymerase

leading strand

the strand used by polymerases as a template to make a new, continuous complimentary strand

run 5' ->3' into the fork

DNA polymerase

lagging strand

100-200 nucleotides long

joined by DNA ligase to form a continuous DNA strand

DNA polymerase

To what can they only add new nucleotides?

1. In order to start a new chain, what does polymerase need?
2. What is primase?

3. What happens after a primer is formed?

1. a primer - a short segment of RNA

about 10 nucleotides long in eukaryotes

2. an RNA polymerase that links together ribonucleotides that are complementary to the DNA template, forming the primer

- can start an RNA chain from a single template strand

3. DNA polymerases can add deoxyribonucleotides to the 3' end of the ribonucleotide chain

Another DNA polymerase later replaces the primer ribonucleotides with deoxyribonucleotides complimentary to the template

DNA polymerase

What does the lagging strand require?

What happens when the primer is formed?

formation of a sequence of new primers as the replication fork preogresses

DNA polymerase adds new nucleotides away from the fork until it runs into the previous Okazaki fragment