DNA Replication and Repair

Label diagram

7 - helicase; 3 leading strand; 6/1 parental strand; 8 lagging strand; 2 RNA primer; 4 DNA pol alpha; 5 topoisomerase; 6 DNA pol delta. Primase makes the RNA primer and DNA Pol alpha uses that RNA primer to make the DNA primer. They are a complex. Topoisomerases (or DNA topoisomerases) are enzymes that participate in the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork.

nucleotide excision repair

As mentioned above, nucleotide excision repair is another process that can be used to correct DNA damage from UV radiation. This process is outlined below A group of enzymes scans the DNA for bulky lesions due to pyrimidine dimers and proceeds to remove the DNA segments containing the lesions. A new DNA strand is synthesized by DNA polymerase using the information on the complementary undamaged strand as a template. The DNA ligase covalently links the adjacent DNA to complete the double-stranded DNA.

base excision repair

Base excision repair is an essential process that repairs many different types of damages, including deaminated bases, oxidized bases, and abasic (AP) sites (loss of a base). If the damage is not repaired, a mutation will result. The base excision repair process is outlined below The Base Excision Repair Process Finding the damage: A glycosylase (in Figure 8, it is referred to specifically as uracil DNA glycosidase) recognizes the mismatch, and cleaves the glycosidic bond between the base and the sugar. Removal of the damage: The base is removed, leaving the backbone intact. The backbone is cleaved by an AP endonuclease and the sugar is removed by an AP lyase. Repair: DNA polymerase extends the DNA segments and DNA ligase covalently links them to each other.

Questions

C

DNA repair systems

D

telomerase

Did you know the length of telomeres is inversely related to the number of times the cell has divided? One of the causes of cellular aging is that normal cells lose their telomerase as they age, resulting in the incomplete ends of DNA. This triggers a damage response that includes cell death. Malignant cells retain the telomerase activity, which may contribute to their ability to survive and replicate indefinitely. Nearly all human cancers studied thus far exhibit high telomerase activity.

DNA polymerases

In humans, the replicative DNA polymerases are called DNA polymerase delta (lagging) and epsilon (leading). DNA polymerase I is a prokaryotic DNA polymerase.

sources of DNA damage

Ionizing radiation and certain chemicals can cause double strand breaks - in addition, fail to correct before replication

photoreactivation

One of the most damaging effects on DNA is produced by ultraviolet (UV) light. When UV light strikes DNA, it causes a covalent linkage between adjacent pyrimidine bases resulting in a pyrimidine dimer, often a thymine dimer as shown in the schematics below (covalent bond). The presence of these dimers leads to a bulge in the DNA chain that can interfere with subsequent DNA replication and transcription. The cell has two mechanisms of removing the pyrimidine dimers. One is nucleotide excision repair, which will be discussed in next section of this handout. The other is Photoreactivation, which is the direct reversal of the damage by photoreactivation by DNA photolyase. As shown in the schematic below, the steps to this process are: a) Binding of DNA photolyase to the pyrimidine dimer b) Absorption of light by the complex resulting in cleavage of the bonds linking the adjacent pyrimidines c) Dissociation o DNA photolyase from the DNA

case 2 - 45 y/o male with blood in stool, colonoscopy removes several polyps of varying sizes from distal colon, pathology report showed malignant transformation in two of the polyps; multiple family members have variety of cancers, including colon cancer and uterine cancer, requests genetic testing

D. 60-70% of HNPCC cases are results of mutations in hMSH2 or hMLHI that encode proteins of the methyl-directed mismatch repair system --> unrepaired mutations --> unrestrained cell growth --> tumors. specifically, a gene called hMSH2 is longer than that from a normal individual, has increased in the lenghts of dinucleotide repeats, which affects the DNA mismatch repair system

methyl-directed mismatch repair

DNA polymerase has a proof-reading capability to correct the mismatch during replication. If the DNA polymerase fails to correct the mismatch during the replication process, there is a "window of opportunity" for the first few hours immediately after replication to repair the mismatch. The opportunity is due to the fact that the A base in the GATC sequences in the DNA usually becomes methylated after several hours following completion of replication. Therefore, a newly replicated DNA molecule is transiently hemi-methylated, i.e., for a limited times the parental DNA strand is methylated whereas the daughter strand is not. The Repair Process Finding the mismatch: A group of proteins and enzymes "scan" the newly synthesized DNA, looking for the bulges that a mis- match produces. Removal of the mismatch: The methyl groups on the parental strand direct the enzymes to remove the DNA on the strand in which the mismatched nucleotide is found. Repair: After removal of the DNA strand containing the mismatch, a gap is created. DNA polymerase then synthesizes a new DNA segment to fill the gap and DNA ligase catalyzes formation of a phosphodiester bond covalently linking the new DNA to the remaining DNA.

initiation: key players and definitions

DNA topoisomerase, binding proteins, helicase

10 y/o child with very fair skin tone, very susceptible to sun burns, examination of extremely dry skin, numerous freckles, presence of several skin lesions with irregular shapes, eyes bloodshot

E. xeroderma pigmentosum disease due to defect in repair of thymidine dimers resulted from UV exposure in skin cells (keratinocytes) due to mutations in genes encoding components of the nucleotide excision repair pathway. clinical symptoms: extremely sensitive to UV light leading to severe sunburn, ultra-dry skin and blisters, skin tumors. persistence of damaged DNA --> replication leads to new DNA with mutations --> mutated DNA mediates carcinogenesis --> multiple skin tumors. UV rays can damage genes that control cell growth and division - either die or grow too fast/uncontrolled

DNA replication process summary

The double helix is unwound at multiple points. The two DNA strands are separated, which causes the formation of a replication fork. Each individual strand is matched with the complementary base pairs, creating two new double stranded DNA molecules, each of which contains one original and one new strand. The new double stranded DNA molecules are re-coiled into the double helix formation.

nucleotide excision repair slide

XP proteins recognize the damage. also for bulky lesions. NER removes pyrimidine dimers, it is not a mechanism that will remove mismatches. the bulky adduct will be recognized and will be excised and the non-damaged strand will be used as the template for repair. the bulge results from pyrimidine dimers - the other strand will not have pyrimidines at that site, there will be purines - and they will not be dimerized

case 3 - 4 y/o boy complains lacking energy, difficulty walking, does not speak as well as peers, vascular lesions on trunk and neck for last few months, chromosomal analysis reveals increased numbers of gaps compared to a normal individual, increased radiation-induced fragility in cultured cells, is immunodeficient

defects in enzymes that normally repair double-stranded DNA breaks (the gaps). 1% of US population, a primary immunodeficiency disease, affects males and females equally, autosomal recessive, mutated ATM gene. it is progressive, wheelchair-bound by teens, fatal by twenties.

base excision

for example, when guanine becomes oxidized.

elongation: key + define

ligase, primase, polymerase, RNase and FEN1

The Replication Process: Steps in Replication

o Initation o Elongation o Termination

DNA Repair Processes

o Methyl-directed Mismatch Repair o Photoreactivation to Repair Pyrimidine Dimers o Nucleotide Excision Repair o Base Excision Repair

termination

telomeres, telomerase