12 DNA Replication/Recombination

Segment of DNA that undergoes replication

Replicon

Replication starts at the origin and continues until when?

The entire replicon has been made

Form Meselson and Stahl’s Experiment what type of replication does DNA undergo?

Semiconservative

How many bands of DNA would be expected after 2 rounds of replication if it were conservative replication?

Two bands (one in light and one in heavy; never intermediate)

Point at which a double-stranded DNA molecule separates into two single strands that serve as templates for replication

Replication Fork

Two replication forks

Bidirectional

One replication fork

Unidirectional

Which type of replication requires a break in the nucleotide strand to get started?

Rolling circle replication

• initiated by a break in one of the nucleotide strands, which exposes a group and a 3’OH and a 5’-phosphate group

What are the requirements for Replication?

1. template consisting of single-stranded DNA

2. Raw materials (substrates) to be assembled into a new nucleotide strand (dNTPs)

3. Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule

Sugar and nitrogenous base

Nucleoside

Sugar, nitrogenous base, and phosphate

Nucleotide

The raw materials/building blocks that build DNA molecules

• nucleotide consisting of deoxyribose sugar, base, and 3 phosphate groups

• 2 phosphates are cleaved off when the last nucleotide binds its 3’-OH with the 5’-phosphate of the incoming nucleotide

Deoxyribonucleoside triphosphates (dNTPs)

Stages of Replication

• Initiation

• Unwinding

• Elongation

• Termination

A single origin of replication

oriC

In bacteria, what must occur first before the start of any unwinding?

Initiator proteins must bind to the oriC first and create that short section of DNA to be unwound. After this, then helicase can continue to unwind the DNA

What direction does helicase move in bacteria and on what strand?

Moves in the direction of unwinding of replication fork. Is placed on the lagging strand but does not move in the direction of the lagging strand, it moves in the direction that the replication fork is moving which is typically opposite of the lagging strand

What is needed in order for DNA polymerase to start DNA synthesis?

a 3’-OH group to get started

Does primase need a 3’-OH group?

No because it’s RNA polymerase

Capable of adding many nucleotides to the growing DNA strand without releasing the template; strand is not let go until full replication is done

High Processivity (ex: DNA Polymerase III)

What prevents DNA Polymerase III from sliding off the strand and ultimately explains why this enzyme has high processivity?

The β sliding clamp

3’ to 5’ Exonuclease activity is a form of

Proofreading (correction of nucleotides)

Does DNA poly. I or III have lower processivity?

DNA poly I

DNA poly. II,IV, and V

DNA Repair

True or False: DNA polymerase I follows III

True

E.coli method of termination

• blocks movement of helicase and thus stalls unwinding of replication fork

• blocks a replication fork moving in one direction, not both

Tus-Ter Complex

Process by which DNA polymerases remove and replace incorrectly paired nucleotides in the course of replication

Proofreading

Process that corrects mismatched nucleotides in DNA after replication has been completed

• Any incorrectly paired nucleotides remaining after replication produce a deformity in the secondary structure of the DNA is recognized by enzymes

• original DNA is methylated so repair enzymes know which strand to fix

• unmethylated strand is what is fixed

Mismatch Repair

Occurs on the lagging strand where its terminal RNA primer is not located at the end of the chromosome but rather 70 to 100 nucleotides from the end. Once this primer is removed and replaced with DNA through DNA polymerase I, the remaining of the chromosome past the 70 to 100 nucleotides is not replicated and cannot be replicated because a primer was not placed there to provide the 3’-OH group. This results in the chromosome to become shorter with each round of replication and gene sequences are eroded and the cell dies

End-Replication Problem

How is the end-replication problem combatted?

Telomeres have telomerase that extend the G-rich 3’ overhang with short repeated TTAGGG sequences. This is long enough for primase, DNA polymerase I and III, and ligase to replicate the chromosome.

Where is telomerase present in?

• single celled eukaryotes

• germ cells (reproductive cells)

• embryonic cells

• proliferative somatic cells (bone marrow, lining of the intestine, etc)

enzyme that restores telomere length

• composed of protein and RNA components complementary to the G rich strand that serves as a template for DNA synthesis

Telomerase

How is the C-rich strand synthesized?

Speculation is that it’s synthesized with DNA polymerase α and an RNA primer; primer is placed on the C-rich strand after G-rich is extended by telomerase so that the C-rich can copy the G-rich in a complementary manner

What is the role of telomerase in cancer?

most cancer cells have active telomerase allowing them to divide without any shortening of their DNA. It is a factor in cancer and is seemingly needed for cancer to develop; not a direct cause

What evidence is led to believe that archaea is more similar to eukaryotes?

• multiple origins of replication

• sequences recognized by initiator proteins are similar sequences found on eukaryotes

• initiator proteins are similar