Lecture 2 - DNA Replication and Repair

Question 1

Origin of replication

Answer 1

Site on a DNA molecule where replication starts

Question 2

Leading strand

Answer 2

The strand of the DNA double helix that is copied in a continuous fashion.

Question 3

Lagging strand

Answer 3

The strand of the DNA double helix that is copied in a discontinuous fashion.

Question 4

Topoisomerase

Answer 4

Class of enzymes that alter the supercoiling of double-stranded DNA.

Question 5

Helicase

Answer 5

Enzyme that separates the strands of a DNA double helix.

Question 6

Okazaki fragment

Answer 6

A short segment of RNA-primed DNA that is synthesized on the lagging strand during DNA replication.

Question 7

Antimetabolites

Answer 7

A drug or substance that is an antagonist to or resembles a normal metabolite and thus interferes with its function, usually by competing for its receptors or enzymes. Antimetabolites used as anticancer agents include 5-fluorouracil (5-FU).

Question 8

Telomerase

Answer 8

Enzyme consisting of RNA and protein components that maintains the ends of chromosomes by adding specific repeat sequences.

Question 9

Polymerase Slippage

Answer 9

DNA polymerase can lose its place in a repetitive sequence tract, and either leaves out or add an inappropriate number of repeat units.

Question 10

Base Excision Repair (BER)

Answer 10

DNA repair process that involves the excision and replacement of a normal base

Question 11

Nucleotide Excision Repair (NER)

Answer 11

DNA repair process involving the excision and resynthesis of a polynucleotide region

Question 12

Homologous Recombination

Answer 12

In the context of DNA repair, a process involving the recombination between homologous double-stranded DNA molecules.

Question 13

Origins of DNA replication (in eukaryotes)

Answer 13

Unlike many prokaryotes and simple eukaryotes like yeast, there is no defined sequence motif that can be used to identify a human origin of
replication. There are human genomic regions that contain origins; however, the exact location of the
origin within these regions can vary.
Origins occur about once every 100-kb per haploid human genome. This corresponds to about 30,000 origins for the human genome. Not all origins are active at the same time. In fact, the activity of origins is dynamic and serves as means of regulating the speed of DNA replication (for some human cells, on the order of 10 hours). For example, dormant origins can be activated when neighboring replication forks are stalled. In addition, different regions of the human genome replicate at different times in S phase with known
correlations to GC-content, gene density, and transcriptional activity.

Question 14

DNA synthesis (leading/lagging strands, Okazaki fragments, exonuclease, polymerase)

Answer 14

Leading strand DNA synthesis proceeds in the 5’-3’ direction from a single RNA primer (8-12 nucleotides long). Lagging strand DNA synthesis
involves multiple priming events and the generation of Okazaki fragments. Okazaki fragments are quite small (100-200 bp) in eukaryotes, such as humans. Although the exact mechanism is still unclear, the FEN1 endonuclease plays a major role in primer removal in human DNA replication.
At least 14 human genes encode DNA polymerases. The major replication enzymes in
humans are DNA polymerase epsilon (leading strand) and delta (lagging strand). Both DNA
polymerases epsilon and delta have 3’-5’ exonuclease activity, which reduces the error rate
from 10-4 to 10-8 per replicated base pair. This is prior to mismatch repair, discussed later in the lecture.

Question 15

Other accessory proteins required for normal DNA replication

Answer 15

These include DNA helicases and topoisomerases and single stranded DNA binding protein.
Collectively these proteins keep the individual strands at the DNA replication fork separated and remove the topological stress generated during DNA replication.

Question 16

Clinical correlates

Answer 16

Irinotecan is a topisomerase inhibitor typically used to treat colorectal cancer.
Oxaliplatin induces DNA damage and is a highly effective component of a combination chemotherapy used to treat testicular cancer.
Microtuble inhibitors interfere with mitosis and chromosome segregation.
5-fluorouracil is a classic example of an antimetabolite. It blocks the activity of thymidylate synthetase and depletes pools of dTTP in the cell. In addition, it can incorporate into RNA and DNA to cause damage. 5-fluorouracil, irinotecan, and oxaliplatin are used as a highly effective
combination therapy for colorectal cancer.
Their differing mechanisms of action
complement each another.

Question 17

Irinotecan

Answer 17

a topisomerase inhibitor typically used to treat colorectal cancer

Question 18

Oxaliplatin

Answer 18

induces DNA damage and is a highly effective component of a combination chemotherapy used to treat testicular cancer.

Question 19

Microtuble inhibitors

Answer 19

interfere with mitosis and chromosome segregation.

Question 20

5-fluorouracil

Answer 20

is a classic example of an antimetabolite. It blocks the activity of thymidylate synthetase and depletes pools of dTTP in the cell. In addition, it can incorporate into RNA and DNA to cause damage.

Question 21

Telomerase

Answer 21

Telomerase is an enzyme involved in replicating the ends of chromosomes. The ‘end replication’ problem arises on the lagging strand when there is no template from which primers can be made for the generation of Okazaki fragments.
Telomerase provides the template required for this priming. Other proteins located near telomeres form the ‘Shelterin’ complex used to prevent the fusion of chromosomes.

Question 22

Telomeres in Health and Disease

Answer 22

Dyskeratosis congenita is caused by
inherited defects in any of several different genes important for the repair or protection of
telomeres. Depending on the mutated gene, the mode of inheritance varies and ranges
from being X-linked to autosomal dominant to autosomal recessive. Bone marrow failure
and increased cancer risk are among the most severe manifestations. Telomerase is active
in most cancer cells even though their telomeres tend to be shorter. There is evidence that
telomere dysfunction plays a key role in the initiation and progression of prostate cancer. It
has been discovered that mutations in the promoter of the TERT gene (encoding the
protein component of telomerase) occur in some melanomas. The role of telomeres in
cancer, heart disease, and aging is still being actively studied.

Question 23

Introduction to Mutations and DNA Damage in the Genome

Answer 23

Uncorrected replication errors occur at about ~10-9
- 10-11 per incorporated nucleotide.
Coding DNA mutations will occur spontaneously with an average frequency of 1.7×10-6
-1.7×10-8 per gene per cell division. During the ~1016 mitoses in an average human lifetime,
each gene will be a locus for about 108
-1010 mutations! However, for any given gene, only a tiny minority of cells will carry a mutation. In many cases, a gene mutation in a somatic cell
will be inconsequential or simply be the mutation may cause lethality for that single cell. At
least in theory, most cells in the body probably carry at least one somatic mutation.

Question 24

Direct Repair

Answer 24

In humans, direct repair acts is involved in repairing (i) broken phosphodiester bonds (nicks) which can result from ionizing radiation through DNA ligase activity and (ii) O6-methylguanine. The MGMT protein repairs O6-methylguanine lesions by stoichiometrically transferring the alkyl group at the O-6 position to a cysteine residue in the
enzyme. This is a suicide reaction and the enzyme is irreversibly inactivated. Loss of MGMT by somatic mutations or epigenetic silencing is associated with increased cancer risk and sensitivity to methylating agents. This is also important since tumors deficient in MGMT activity could be more responsive to certain types of chemotherapy.

Question 25

Excision Repair

Answer 25

There are at least 40 genes involved in excision repair pathways in the human genome. The two different types of excision repair are base
excision repair (BER) and nucleotide excision repair (NER).
BER is used for bases with relatively minor damage. A DNA glycosylase removes the damaged base and produces an AP site (apurinic/apyrimidinic) without a base. The AP site is removed by other enzymes and a DNA polymerase uses the other strand as a template to fill-in the gap.
NER deals with severe types of damage, such as intrastrand cross-links and bases with unnatural
bulky chemical groups. In NER, a segment of DNA containing the damage is excised and replaced by DNA polymerase activity. The size of the patched segment is ~30 nucleotides in length. Cells have coupled one of the transcription complexes with the NER machinery to remove road blocks to transcription. Thus, DNA damage on the transcribed strand is repaired faster than on the non-transcribed strand due to transcription coupled repair.Defects in NER genes are responsible for the autosomal recessive diseases xeroderma pigmentosa and Cockayne syndrome. Both diseases are characterized by extreme
sensitivity to the sun due to UV radiation exposure, especially the skin and eyes.
Xeroderma pigmentosa patients can have an increased incidence of skin cancer. Cockayne
syndrome patients have defects in the transcription-coupled NER repair pathway and also
show microcephaly (circumference of the head is smaller than normal because the brain
has not developed properly or has stopped growing), and growth and mental retardation.

Question 26

Mismatch Repair

Answer 26

Mismatch repair (MMR) corrects 99.9% of DNA
replication errors. The overall mutation frequency is 1 in one billion nucleotides synthesized
in the final product. It is still not clear how the daughter strand is distinguished from the
mother strand, which serves as a template for correcting the mistake. Inherited mutations in
MMR related genes can lead to Lynch
syndrome, aka hereditary nonpolyposis
colorectal cancer (HNPCC), a type of
inherited cancer of the digestive tract,
particularly the colon and rectum. Lynch
syndrome is inherited in an autosomal
dominant manner. Somatic mutations
acquired in one’s adult cells can lead to
‘sporadic’ cases of colorectal cancer.
Tumors cells with MMR defects show microsatellite instability (MSI) wherein specific types of repetitive sequences in the genome can expand or contract in length during DNA
replication. You will be responsible for interpreting the results of PCR-based assays like the ones shown on slide 38. Simply put, if the PCR products from normal and tumorderived tissue differ, there is MSI. The amplified genomic region is chosen to contain repetitive sequences.

Question 27

Nonhomologous End Joining (NHEJ)

Answer 27

Some DSBs are created by ionizing radiation or by oxidative free radicals during metabolism. But many are created during S phase of the cell cycle, when a replication fork encounters a nick. NHEJ can repair a double strand break (DSB) any time during the cell cycle and does not require homologyat the ‘broken ends’, but a few nucleotides of terminal homology are often utilized. NHEJ is imprecise and can be responsible for structural changes in the chromosomes of some
cancer cells, such as translocations, inversions, and deletions.

Question 28

Homologous Recombination (HR)

Answer 28

HR is mechanism for repairing DSBs
that is more precise than NHEJ. HR is highly active during late S and G2 of the cell cycle.
These are phases when sister chromatids in mitosis, and sisters and homologues in meiosis can serve as homology donors because of their close proximity. HR involves regions of DNA homology that are typically >25 base pairs and usually hundreds to thousands of base pairs in length.

Question 29

Fanconi Anemia

Answer 29

Fanconi anemia is primarily an autosomal recessive disorder that is caused by mutations in any of a set of 15 genes involved in DNA repair.
Cells from patients are hypersensitive to DNA cross-linking agents and develop chromosomal aberrations including breakage. Patients show developmental abnormalities in major organ systems, upper limb malformations, early-onset bone marrow failure, and a high predisposition to cancer.