Lecture 2 - DNA Replication and Repair Flashcards
Learning Objectives: 1. Identify therapeutic agents that affect DNA replication and the describe basis for their activity 2. Describe how defects in DNA repair and replication lead to common and rare diseases 3. Name diseases whose pathogenesis is related to inherited or acquired defects in DNA repair and replication 4. Recall the roles of telomere dysfunction in common and rare diseases
Origin of replication
Site on a DNA molecule where replication starts
Leading strand
The strand of the DNA double helix that is copied in a continuous fashion.
Lagging strand
The strand of the DNA double helix that is copied in a discontinuous fashion.
Topoisomerase
Class of enzymes that alter the supercoiling of double-stranded DNA.
Helicase
Enzyme that separates the strands of a DNA double helix.
Okazaki fragment
A short segment of RNA-primed DNA that is synthesized on the lagging strand during DNA replication.
Antimetabolites
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).
Telomerase
Enzyme consisting of RNA and protein components that maintains the ends of chromosomes by adding specific repeat sequences.
Polymerase Slippage
DNA polymerase can lose its place in a repetitive sequence tract, and either leaves out or add an inappropriate number of repeat units.
Base Excision Repair (BER)
DNA repair process that involves the excision and replacement of a normal base
Nucleotide Excision Repair (NER)
DNA repair process involving the excision and resynthesis of a polynucleotide region
Homologous Recombination
In the context of DNA repair, a process involving the recombination between homologous double-stranded DNA molecules.
Origins of DNA replication (in eukaryotes)
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.
DNA synthesis (leading/lagging strands, Okazaki fragments, exonuclease, polymerase)
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.
Other accessory proteins required for normal DNA replication
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.
Clinical correlates
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.
Irinotecan
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.
Telomerase
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.
Telomeres in Health and Disease
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.
Introduction to Mutations and DNA Damage in the Genome
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.
Direct Repair
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.
