cell bio chapter 6 - DNA replication and repair

DNA replication

accurate duplication of the genetic information carried in DNA

When does DNA replication occur?

before a cell can produce 2 genetically identical daughter cells

Mutations

changes of the DNA caused by copying errors and accidental damage

Replication machinery

cluster of proteins that carry out DNA replication

When is a DNA double helix opened at?

The replication origin

Initiator proteins

recognize sequences of DNA at replication origins and locally pry apart the two strands of the double helix

What is origin licensing?

The process in G1 where proteins assemble at DNA replication origins, marking them as ready for replication so each origin fires only once per cell cycle.

What does the ORC do?

Recognizes and binds to the origin of replication, acting as the starting platform for assembling the proteins needed to license DNA replication.

MCM complex

Component of helicase that unwinds the DNA double helix so it can be copied

MCM cycle

Highly regulated and essential to license origins in only G1

What does the release of CDT1 do?

allows the helicase (CMG complex) to fire the origin

How do replication forks move?

away in opposite directions from multiple replication origins

What direction is DNA synthesized?

5’ to 3’

nucleoside triphosphates

building blocks used to make RNA.

DNA polymerase

Adds nucleotides to the 3′ end of a growing DNA strand

What does breaking a phosphoanhydride bond do?

it releases a large amount of free energy and thus provides the energy for the
polymerization reaction

Lagging strand

DNA strand that’s synthesized discontinuously

okazaki fragments

Short pieces of new DNA made discontinuously on the lagging strand

How does DNA polymerase proofread?

If an incorrect nucleotide is added to a growing strand, it will cleave it and replace it with the correct nucleotide

What are DNA’s polymerase’s separate sites for?

for DNA synthesis and proofreading

How is DNA synthesized on the lagging strand?

In fragments

RNA primers

Made at intervals of about 200 nucleotides on the lagging strand by primase

Nucleases

remove primers by recognizing an RNA strand in the helix and degrading it

DNA ligase

joins together Okazaki fragments and catalyzes the formation of a phosphodiester bond

What does the nick-sealing reaction need?

an input of energy in the format of ATP or NADH

What direction does primase synthesize primers?

5’ to 3’

DNA polymerase vs RNA polymerase/primase

Unlike DNA polymerase, primase can start a new polynucleotide chain on its own

single strand binding proteins (RPA)

Bind to and stabilize separated single-stranded DNA during replication

PCNA

A circular sliding clamp that holds DNA polymerase onto DNA and allows it to slide along the DNA during replication.

CMG helicase

Uses ATP hydrolysis to move along DNA and separate the parental DNA strands at the replication fork

How are the leading and lagging strand DNA polymerases positioned relative to each other?

The lagging strand folds so Pol δ is positioned near Pol ε on the leading strand.

What does folding the lagging strand accomplish for Okazaki fragments?

It brings the 3′ end of the completed Okazaki fragment close to the start site of the next Okazaki fragment.

Why can Pol (delta) δ synthesize multiple Okazaki fragments?

The folded lagging strand brings each new fragment's start site close to Pol (delta) δ, allowing the same polymerase to be reused

DNA topoisomerases

Enzymes that relieve torsional stress ahead of the replication fork by making temporary DNA nicks that allow rotation and release of tension

torsional stress

Tension caused by DNA overwinding as helicase unwinds the double helix

supercoils

Additional coiling of DNA that forms in response to torsional stress.

DNA topoisomerase I

Temporarily cuts one DNA strand, allowing the DNA to rotate and release torsional stress

DNA topoisomerase II

Temporarily cuts both DNA strands, allowing a section of DNA to pass through and relieve torsional stress

clamp loader

uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA

FISH

used for visualizing telomeres

telomeres

Repetitive DNA sequences at the ends of chromosomes that protect chromosome ends.

G-overhang

A single-stranded DNA overhang at the end of a telomere that binds telomere-protecting proteins.

Telomere end replication problem

The difficulty of fully replicating the ends of linear chromosomes, causing telomeres to shorten with each round of DNA replication

Shelterin complex

Proteins that bind to telomeres and protect chromosome ends from being recognized as DNA damage

telomerase

Enzyme that extends the 3′ end of the template DNA by adding repeated DNA sequences, allowing the lagging strand to be fully replicated.

Telomerase RNA

Short RNA sequence within telomerase that serves as a template for adding telomeric DNA repeats.

Telomere extension process

adds DNA repeats to the 3′ end of the template strand, providing enough template for DNA polymerase to complete the lagging strand.

beta globin

A subunit of hemoglobin

sickle cell mutation

Single nucleotide change in the β-globin gene

Sickle-cell inheritance

One mutant copy generally causes no harm

Two mutant copies cause sickle-cell anemia

Cancer and age

Cancer incidence increases with age because mutations accumulate over time

Uncorrected mismatch

Becomes a permanent mutation after the next round of DNA replication

mismatch repair

Uses the parental DNA strand as the template to correct the newly synthesized strand

DNA mismatch repair proteins

Correct errors made during DNA replication

Newly synthesized DNA strand

Nicked during mismatch repair to identify the strand that needs repair

depurination

Loss of a purine base from DNA

purines

adenine (A) and guanine (G)

deamination

removal of an amino group from cytosine → produces uracil (U)

UV radiation

Causes DNA damage by creating thymine dimers

thymine dimer

Two adjacent thymine bases become covalently bonded together

3 steps of DNA repair

excision, resynthesis, ligation

excision

Nucleases remove the damaged DNA

resynthesis

Repair DNA polymerase fills the gap with the correct DNA sequence

ligation

DNA ligase seals the nick in the sugar-phosphate backbone using ATP energy

single nucleotide repair

Some damage such as cytosine deamination requires replacement of a single nucleotide

thymine dimer repair

Requires removal and replacement of a longer stretch of about 10–20 nucleotides

Homologous recombination

Most accurate form of double-strand break repair

What are some limitations of homologous recombination?

it uses identical sister chromatid as a template so it is mainly possible after DNA replication

Non-homologous end joining

Directly joins the broken ends. Some DNA may be lost or added

NHEJ mechanism

Directly joins the two broken DNA ends without using a template

Why is NHEJ error-prone?

DNA ends may be trimmed or altered before being joined, causing loss or addition of nucleotides

Alternative end joining

error-prone mechanism for repairing double-strand breaks

doesn’t require extensive homology and a sister chromatid is not required

Alt-EJ mechanism

Uses short regions of matching DNA to align broken DNA ends

homology

Similarity or matching between DNA sequences