Unit 10: DNA Replication and Repair

Describe the orientation of DNA strands in the double helix

Two polynucleotide chains run in opposite directions (antiparallel) and are held together by hydrogen bonds

Describe the structure of the DNA double helix

The strands coil around a common axis to form a double helix

Describe the location of components of the DNA helix

Bases are on the inside, and the sugar-phosphate backbone is on the outside

Describe base pairing in DNA

A pairs with T (2 hydrogen bonds) and C pairs with G (3 hydrogen bonds), each consisting of one purine and one pyrimidine

Describe β-DNA

The standard β-helical form of DNA that is determined by X-ray diffraction

Describe the rotational flexibility of DNA

DNA can rotate around about six bonds, including two glycosidic bonds and four phosphodiester bonds

Describe the planarity of base pairs

Base pairs do not have to be perfectly coplanar

Describe ways DNA structure can change shape

DNA can bend into an arc, supercoil, wrap around proteins (histones), or form kinks at discrete sites

Describe why DNA structural variability is important

It allows proteins to recognize and bind specific DNA sequences

Describe how DNA structure affects gene regulation

Changes in structure can upregulate or downregulate gene expression

Describe another functional role of DNA flexibility

It allows DNA to compact and fit inside the cell

Describe how major and minor grooves are formed in DNA

They arise because the glycosidic bonds of a base pair are not directly opposite each other

Describe the chemical properties of DNA grooves

Each groove contains hydrogen bond donors and acceptors

Describe the difference between major and minor grooves

The major groove is larger and more accessible for protein binding

Describe semiconservative DNA replication

Each new DNA molecule contains one original strand and one newly synthesized strand

Describe the role of each DNA strand during replication

Each strand serves as a template for the synthesis of a new complementary strand

Describe the origin of replication (ORI)

A specific site where DNA replication begins

Describe the direction of DNA synthesis

DNA is synthesized in the 5′ → 3′ direction

Describe the concept of semi-discontinuous replication

One strand is synthesized continuously, while the other is synthesized in fragments

Describe the leading strand

It is synthesized continuously in the same direction as the replication fork

Describe the lagging strand

It is synthesized discontinuously in short segments opposite the direction of the replication fork

Describe okazaki fragments

Short DNA fragments that are formed during lagging-strand synthesis

Describe bidirectional DNA replication

Replication proceeds in both directions from the origin

Describe nucleases (DNases)

Enzymes that degrade DNA

Describe exonucleases

They remove nucleotides from the ends of DNA strands and are essential for DNA replication

Describe endonucleases

They cleave DNA at specific internal sites within a strand

Describe DNA polymerase

An enzyme that synthesizes DNA using a template strand

Describe DNA Polymerase I

- The first DNA polymerase isolated from E. coli that functions as a template-directed enzyme

- Template-directed enzyme in which elongation proceeds in the 5' to 3' direction

Describe how DNA polymerases synthesize DNA

They add nucleotides in the 5′ → 3′ direction using a template strand

Describe the role of the 3′-OH group in DNA synthesis

The 3′-OH acts as a nucleophile to attack the incoming nucleotide

Describe the role of Mg²⁺ in DNA polymerase activity

Mg²⁺ ions help stabilize charges and assist in catalysis

Describe the role of incoming dNTPs in DNA synthesis

They provide nucleotides and release energy through pyrophosphate cleavage

Describe the energy source for DNA polymerization

Energy comes from the release of pyrophosphate (PPi)

Describe a primer in DNA replication

A short RNA oligonucleotide complementary to the DNA template

Describe the function of a primer in DNA replication

It provides a free 3′-OH group (primer terminus) for DNA synthesis to begin

Describe how primers are synthesized

They are made by specialized enzymes (primase)

Describe the fidelity of primers compared to DNA polymerase

Primers have lower fidelity but are temporary and later removed

Describe the accuracy of DNA replication

DNA replication is extremely accurate

Describe how correct nucleotides are selected during replication

Selection depends on complementary base pairing and proper base pair geometry

Describe complementary base pairing in DNA replication

A pairs with T and G pairs with C based on Watson-Crick rules

Describe how DNA polymerase I selects correct nucleotides

The active site of DNA polymerase I only accommodates nucleotides with the correct base pair geometry

Describe why incorrect nucleotides are rejected by DNA polymerase

They do not properly fit into the active site even if hydrogen bonds can form

Describe how abnormal base pairs interact with the DNA polymerase active site

They sit outside the normal binding pocket and do not fit correctly

Describe the proofreading function of DNA polymerase

All DNA polymerase have 3′ → 5′ exonuclease activity that removes incorrectly added nucleotides

Describe how DNA polymerase ensures replication accuracy after nucleotide addition

It double-checks each nucleotide using exonuclease activity

Describe the relationship between proofreading and polymerization

Proofreading is a separate function and not simply the reverse of polymerization

Describe the role of pyrophosphate (PPi) in DNA synthesis

PPi is hydrolyzed after nucleotide addition, driving the reaction forward

Describe why DNA polymerization is effectively irreversible

Pyrophosphate (PPi) hydrolysis creates a large negative ΔG

Describe how incorrect base pairing can still occur during replication

Tautomeric shifts can alter hydrogen bonding patterns

Describe an example of abnormal base pairing

Cytidine can pair with thymidine due to tautomeric changes

Describe the first step of DNA polymerase proofreading

A mismatch is formed when an incorrect nucleotide is incorporated

Describe how DNA polymerase responds to a mismatch

The enzyme pauses and shifts the 3′ end of the DNA to the exonuclease site

Describe the role of the 3′ → 5′ exonuclease site

It removes incorrectly paired nucleotides

Describe how the incorrect nucleotide is removed

The exonuclease hydrolyzes and releases the mismatched nucleotide

Describe what happens after the incorrect nucleotide is removed

The corrected 3′ end returns to the polymerase active site

Describe how DNA synthesis resumes after proofreading

DNA polymerase adds the correct nucleotide and continues elongation

Describe the purpose of proofreading in DNA replication

It increases the accuracy of DNA synthesis

Describe proofreading as a quality control step

It acts as a second check after base pairing (with the template being the first check)

Describe the selectivity of proofreading activity

It can remove some correctly paired nucleotides as part of error checking (1 in 10 correctly paired nucleotide)

Describe the 5′ → 3′ exonuclease activity of DNA Polymerase I

A function that removes nucleotides ahead of the polymerase during DNA synthesis

Describe the requirement for cleavage by the 5′ → 3′ exonuclease

The DNA must be in a double-helical region, not a free strand

Describe how DNA synthesis affects 5′ → 3′ exonuclease activity

The activity is enhanced when DNA synthesis is occurring simultaneously

Describe nick translation

A process where DNA Polymerase I removes a segment of DNA or RNA and replaces it with new DNA

Describe the role of nick translation in replication

It replaces RNA primers with DNA

Describe one primary function of DNA Polymerase I

Removal of RNA primers

Describe another primary function of DNA Polymerase I

Repair of DNA errors or damage

Describe how DNA Polymerase I removes nucleic acids during nick translation

It cleaves nucleotides ahead of the nick while synthesizing new DNA behind it

Describe how nucleotides are replaced during this process

dNTPs are added while released nucleotides (NMPs or dNMPs) are removed

Describe how the nick moves during nick translation

The nick shifts forward as DNA Polymerase I removes and replaces nucleotides

Describe the final outcome of nick translation

The RNA primer or damaged DNA segment is replaced with newly synthesized DNA

Describe DNA polymerases II and III

They are similar to DNA polymerase I and catalyze template-directed DNA synthesis

Describe the primer requirement for DNA polymerases II and III

They require a primer with a free 3′-OH group

Describe the direction of DNA synthesis by DNA polymerases II and III

They synthesize DNA in the 5′ → 3′ direction

Describe the proofreading ability of DNA polymerases II and III

They possess 3′ → 5′ exonuclease activity

Describe the primary role of DNA polymerase III

It synthesizes most of the new DNA

Describe the primary role of DNA polymerase I

It removes RNA primers and replaces them with DNA

Describe the role of DNA polymerase II

It participates in DNA repair

Describe oriC

The origin site where DNA replication begins in E. coli

Describe a key feature of the oriC sequence

It is enriched in 5' GATC sequences

Describe the role of DnaA protein

It recognizes oriC and opens the DNA duplex

Describe methylation at oriC

Dam methylase methylates the N6 of adenosine within GATC sequences

Describe the purpose of DNA methylation at oriC

It distinguishes between parent and daughter strands

Describe the function of DnaB (helicase)

It unwinds double-stranded DNA into single strands

Describe the function of primase

It synthesizes RNA primers

Describe the function of SSB proteins

They bind single-stranded DNA and stabilize it

Describe the function of DNA gyrase

It relieves strain and alters DNA conformation during unwinding

Describe the role of DNA polymerase III

It synthesizes new DNA during replication

Describe the role of DNA polymerase I

It removes RNA primers and fills the gaps with DNA

Describe a replication fork

A site where DNA is unwound and replicated at the same time

Describe how DNA synthesis relates to DNA unwinding

DNA synthesis is coupled to the unwinding of parental DNA

Describe whether DNA is fully unwound before replication begins

DNA is not completely unwound prior to replication

Describe the direction of DNA synthesis at the replication fork

DNA is synthesized in the 5′ → 3′ direction

Describe how the leading strand is synthesized

It is synthesized continuously in the direction of the replication fork

Describe how the lagging strand is synthesized

It is synthesized discontinuously in short fragments

Describe Okazaki fragments

Short DNA segments formed on the lagging strand

Describe the role of helicase (DnaB)

It unwinds the DNA double helix

Describe the role of primase

It synthesizes RNA primers

Describe the role of SSB proteins

They stabilize single-stranded DNA

Describe the role of DNA gyrase (topoisomerase II)

It relieves strain caused by DNA unwinding

Describe how both strands are synthesized simultaneously

The lagging strand loops to allow coordinated synthesis with the leading strand