Bio 310 Chapter 9: DNA Replication and Recombination

A single-celled human zygote contains _______ base pairs of DNA

6.4 billion

Semi-Conservative replication

Replication is when the two nucleotide strands of DNA separate, and each serves as a template for the synthesis of a new complementary strand. All DNA replication is semiconservative.

Equilibrium density gradient centrifugation

Method used to separate molecules or organelles of different density by centrifugation.

What were the three models initially proposed for DNA replication

conservative, semi-conservative, and dispersive replication.

Replicon

Unit of replication consisting of DNA from the origin of replication the point at which replication on either side of the origin ends.

Origin of Replication

Site where DNA replication is initiated

Theta Replicatoin

Replication of circular DNA that is initiated by the unwinding of the two nucleotide strands, forming a replication bubble. Unwinding continues at one or both ends of the bubble, making it progressively longer. DNA replication on both of the template strands is simultaneous with unwinding until the two replication forks meet.

Replication Bubble

The structure formed during DNA replication where the double helix unwinds and two replication forks are created, allowing for the synthesis of new DNA strands.

Bidirectional replication

A mode of DNA replication where two replication forks move in opposite directions from a single origin, allowing for simultaneous synthesis of new strands.

Both bidirectional and unidirectional replication produce…

Two complete circular DNA molecules, each consisting of one old and one new nucleotide strand.

How do large linear chromosomes, like those found in eukaryotic cells, replicate so quickly?

Replication is initiated at thousands of origins. This allows for multiple replication bubbles to form, enabling simultaneous synthesis of DNA across the chromosome.

Although the process of replication includes many components, they can be combined into 3 major groups, what are they?

1. A template consisting of single-stranded DNA

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

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

DNA Polymerases

Enzyme that synthesizes DNA

Continus Replication

Replication of the leading strand of DNA in the same direction that of unwinding, allowing new nucleotides to be added continuously to the 3’ end of the new strand as the template is exposed.

Leading strand

DNA strand that is replicated continuously

Discontinus Replication

Replication of the lagging strand of DNA in the direction opposite that of unwinding, which means that DNA must be synthesized in short segments called Okazaki fragments.

Lagging Strand

DNA strand that is replicated discontinuously

Okazaki Fragments

Short segments of newly synthesized DNA produced during the discontinuous replication of the lagging strand. These fragments are eventually joined together.

The four stages of DNA replication

1. Initiation

2. Unwinding

3. Elongation

4. Termination

Initiator Proteins

Protein that binds to an origin of replication causes a short section of DNA to unwind, allowing helicase and other proteins to attach to the polynucleotide strand.

what occurs during the Initiation step of DNA replication

Initiator proteins bind to the origin of replication, unwinding a small section of the DNA. Helicase and other single-strand binding proteins can then attach to the polynucleotide strand.

what occurs during the unwinding step of DNA replication

The DNA double helix is separated into two single strands by helicase, allowing the replication machinery to access the template strands for synthesis.

DNA Helicase

Enzyme that unwinds double-stranded DNA by breaking hydrogen bonds that exist between the bases of two nucleotide strands of a DNA molecule.

Single-strand binding proteins (SSBs)

Proteins attach tightly to the exposed single-stranded DNA during replication and prevent the formation of secondary structures that could interfere with the replication process.

DNA Gyrase

Topoisomerase enzyme in E. coli which relieves the torsional strain that builds up ahead of the replication fork.

Primase

Enzyme that synthesizes a short stretch of RNA on a DNA template; provides a 3’-OH group for attachment of a DNA nucleotide at the initiation of replication.

Primers

Short stretch of RNA on a DNA template; functions in replication to provide a 3’-OH group for attachment of a DNA nucleotide.

DNA polymerase III

Bacterial DNA polymerase that synthesizes new nucleotide strands by adding new groups to the 3’-OH group provided by the primer

DNA polymerase I

Bacterial DNA polymerase that removes RNA nucleotides of the primers and replaces them with DNA nucleotides

Despite all of their differences, e. coli’s DNA polymerases have these things in common:

1. synthesize any sequence specified by the template strand.

2. synthesize in the 5'→3' direction by adding nucleotides to a 3'-OH group.

3. use dNTPs to synthesize new DNA.

4. require a 3'-OH group to initiate synthesis.

5. catalyze the formation of a phosphodiester bond by joining the 5'-phosphate group of the incoming nucleotide to the
3'-OH group of the preceding nucleotide on the growing strand, cleaving off two phosphates in the process.

6. produce newly synthesized strands that are complementary and antiparallel to the template strands.

7. are associated with a number of other proteins.

DNA Ligase

Enzyme that catalyzes the formation of a phosphodiester bond between adjacent 3’-OH and 5’-phosphate groups in a DNA molecule without adding another nucleotide to the strand.

Each active replication fork requires these 5 basic components

1. Helicase to unwind the DNA

2. Single-strand binding proteins to protect the single nucleotide strands and prevent secondary structure

3. DNA gyrase to remove strain ahead of the replication fork

4. primase to synthesize primers with a 3’-OH at the beginning of each DNA fragment

5. DNA polymerase to synthesize the leading and lagging nucleotide strands.

Proofreading

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

Mismatch Repair

Process that corrects mismatched nucleotides in DNA after replication has been completed. Enzymes excise incorrectly paired nucleotides from the newly synthesized strand and use the oiginal nucleotide strand as the template for replacing them

Replication Licensing Factors

Protein that ensures the replication takes place only once at each origin of replication; required at the origin before replication can be initiated and removed after the DNA has been replicated.

DNA Polymerase α

Eukaryotic DNA polymerase that initiates nuclear DNA synthesis.

DNA Polymerase δ

Eukaryotic DNA polymerase that replicates the lagging strand during DNA synthesis; also carries out DNA repair and translesion DNA synthesis.

DNA Polymerase ε

Eukaryotic DNA polymerase that replicates the leading strand during DNA synthesis.

Translesion DNA polymerases

Specialized DNA polymerase that is able to replicate DNA through distorted structures and bulky lesions that halt other DNA polymerases; often makes more errors during DNA synthesis than other DNA

Telomerase

Ribonucleoprotein enzyme that replicates the ends (telomeres) of eukaryotic chromosomes. The RNA part of the enzyme has a template that is complementary to repeated sequences in the telomere and pairs with them, providing a template for the synthesis of additional copies of repeats.

Homologous recombination

Exchange of genetic info between homologous DNA molecules

Heteroduplex DNA

DNA consisting of two strands, each of which is from a different chromosome.

Holliday Junction

Special structure resulting from homologous recombination that is initated by single-strand breaks in a DNA molecule.