DNA Metabolism and DNA Replication 1

Is DNA’s structure dynamic or static

Dynamic

What influences DNA’s dynamic structure

DNA Replication

Does DNA replication occur with high or low fidelity before each cell division

High

What happens to errors that occur during or after replication

They are recognized and corrected

How can DNA segments arrange themselves and what does it contribute to

Either within or between DNA molecules, contributing to genetic variation in offspring

Is DNA replication template dependent

Yes, one strand serves a the template for synthesizing its complement

Watson-Crick base pairing rules

Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G)

At what site does new DNA synthesis occur, when nucleotides are added to the growing DNA strand

the 3’ hydroxyl (OH) end

What mediates DNA synthesis

DNA polymerase

DNA polymerase function

Catalyze the addition of nucleotides by forming a phosphodiester bond between the 3’ OH of the growing strand and the a-phosphate of an incoming deoxynucleotide triphosphate (dNTP)

What is released during nucleotide addition

Pyrophosphate (PPi)

Does pyrophosphate (PPi) release drive the reaction energetically forward or backward

Forward

What did the Meselson-Stahl experiment show

It showed that the nitrogen used for the synthesis of new dsDNA becomes equally divided between the two daughter genomes

What did we learn from the Meselson-Stahl experiment

DNA replication is semiconservative

Describe the Meselson-Stahl experiment:

•Cells were grown on a medium containing only ¹⁵N isotope until all their DNA became fully ¹⁵N labeled

•Cells were then switched to ¹⁴N medium and allowed to divide once

• The resulting DNA had intermediate density, proving each daughter DNA molecule contains one old (parental) and one

What gradient was used to analyzed the density of the DNA strands

A cesium chloride (CsCl) density gradient centrifugation was used to determine the mass before and after each round

What does DNA Polymerase I aid in the cell

Cleaning during replication, repair, and recombination

What is the most abundant polymerase

DNA polymerase I

What does DNA polymerase II aid in

DNA repair

What does DNA polymerase III aid in

DNA replication

What polymerase is the main enzyme for DNA synthesis

DNA polymerase III

What ion is required by DNA polymerases to coordinate the a-phosphate, and what function does it serve

Mg²⁺ ions are required to stabilized the transition state during catalysis

What does the 3’ —> 5’ exonuclease activity of DNA polymerases ensure

High fidelity by excising incorrectly paired nucleotides during synthesis

What direction does DNA synthesis proceed

In the 5’ to 3’ direction

Is leading strand synthesis continuous or discontinuous with the replication fork’s progression

Continuous

Is lagging strand synthesis continuous or discontinuous with the replication fork’s progression

discontinuous

What is produced on the lagging strand as a result of it’s discontinuous nature

Okazaki fragments (short DNA fragments)

What are Okazaki fragments initiated by

RNA primers

What enzyme synthesizes RNA primers needed to initiate DNA synthesis

Primase

What enzyme extends Okazaki fragments

DNA polymerase

What happens to RNA primers later on through the actions of DNA polymerase I

They are removed and replaced by DNA

What seals the nicks between Okazaki fragments, forming a continuous strand

DNA ligase

What is the complete replication machinery known as

The replisome

Components of the replisome

Helicases, topoisomerases, single-strand DNA-binding proteins (SSB), primases, and DNA ligases

Helicase function

Use ATP hydrolysis to unwind the DNA double helix

Topoisomerase

Prevents supercoiling and relieves topological stress during strand separation

Single-strand DNA-binding proteins (SSB)

Stabilize separated DNA strands to prevent reannealing

DNA ligase

Seal phosphodiester backbone nicks in DNA

What are the stages of DNA replication

Initiation, Elongation, Termination

Initiation stage

Occurs at specific origins of replication, involving multiple proteins and is tightly regulated to ensure replication happens once per cell cycle

Elongation stage

Leading strand is synthesize continuously; lagging strand synthesized discontinuously in Okazaki fragments. RNA primers are synthesized and later replaced by DNA

Termination stage

Replication forks meet at termination sites where Tus protein binds to arrest fork progression

What does correcting errors in DNA sequence do

Maintain genetic integrity

How are errors in DNA sequences corrected

Proofreading activity of DNA polymerases (3’ —> 5’) and Post-replication repair systems

Is DNA repair template dependent

Yes, it is needed to correct mismatches in the daughter strand

Homologous recombination

The exchange of DNA segments between molecules with similar sequences, contributing to genetic diversity and repair

What degradation enzymes are involved in DNA/RNA and what do they degrade

Nucleases (nucleic acids), DNase (DNA), RNase (RNA)

What cleavage enzymes are involved in DNA/RNA and what do they cleave

Exonuclease (cleaves nucleotides one at a time from an end of DNA or RNA), and Endonuclease cleaves nucleotides at internal sites within DNA or RNA

Do DNA viruses encode their own DNA polymerases

Yes, they are distinct from host enzymes

What are targets for antiviral drugs

Viral DNA polymerases