DNA Damage and Repair (#1)

Base Excision Repair

A. The damaged site (blue dot) is recognized by the repair enzymes. B. and C. Repair enzymes remove the altered/damaged nucleotide in a step-wise fashion. D. The single nucleotide gap is filled by a DNA polymerase. E. The remaining nick is sealed by a DNA ligase (ligase = to tie or join).

Nucelotide excision repair

A. The damaged site, a thymine dimer in this case (blue rectangle), is recognized by the repair enzymes. B. and C. Repair enzymes remove a section of DNA that contains the damaged nucleotide. D. The gap is filled by a DNA polymerase. E. The remaining nick is sealed by a DNA ligase (ligase = to tie or join).

Recombination Repair of a DNA double-strand break.

A. The double-strand break pieces (orange) are aligned with homologous regions (outlined in green) of undamaged DNA (blue) - usually from the second copy of the same chromosome. B. The strands of the undamaged DNA are used by several enzymes to help provide the needed sequence for the damaged DNA. C. As a result of the repair process, some of the sequence from each set of DNA strands has been "mixed".

An alteration in a cell's DNA:

An alteration in a cell's DNA, whether from a poly-merase error or another cause, becomes a permanent mutation unless it is repaired. A mutation can affect the expression of genes positively or negatively (or it may have no measurable effect on the organism). The accumulation of genetic changes over an individual's lifetime may contribute to the gradual loss of functionality associated with aging.

What are mutations?

Are inheritable changes in the DNA sequence.

Mis-sense mutations

Are point mutations that change a single base pair in a codon such that the codon now encodes a different amino acid (Figure 1-10 a).

Non-sense mutations

Are point mutations that change a single base pair in a codon to a stop codon that terminates translation (Figure 1-10 b).

Silent mutations

Are point mutations that result in no change in amino acid sequence due to some redundancy in the genetic code. For example, the codon UCU could have point mutations to UCC, UCA, or UCG and still code for the same amino acid, serine.

Homologous Recombination and Non-homologous end-joining.

Are used to repair double-stranded DNA breaks.

Mutated Genes Can Cause Disease

As discussed in the chapter on Genes to Proteins, the DNA sequence of a gene determines the resulting protein sequence. Therefore, changes to the DNA sequence, whether from damage or replication mistakes by DNA polymerase, can lead to changes in the protein sequence. Sometimes, these changes result in disease. For example, the variant hemoglobin protein that causes sickle cell anemia results from a point (missense) mutation that changes the normal GAG codon to GTG, as shown below. This results in the substitution of the amino acid glutamate with the amino acid valine with serious consequences for those who carry this change in their DNA. (See figure page 3)

What are the two major modes of excision repair?

Base excision repair (BER) and nucleotide excision repair (NER).

What happens when a mis-match is recognized?

But when a mismatch is recognized, both bases are normal; which one should be removed? In newly replicated DNA, removing the newly synthesized base would preserve the genetic information, ------- whereas removing the base from the parental strand would permanently alter the DNA sequence, producing a mutation.-------- Luckily, the cell has reliable ways to distinguish newly synthesized DNA from the parental DNA and reliably remove the erroneous base.

Enzymes

Can recognize damaged bases specifically and remove them, either individually or as part of an oligonucleotide.

Cells with extensive DNA damage:

Cells with extensive DNA damage will escape the normal growth-control mechanisms and proliferate excessively, resulting in cancer. For this reason, cancer can be considered a disease of the genes.

Homologous Recombination.

Certain types of irradiation, such as exposure to X-rays or radioactivity, can completely sever the strands of a DNA double helix, leading to a double-strand break. These breaks, and other types of severe damage, can be re-joined through a process of recombination, called homologous recombination.

Mismatch Repair pathways

Correct mismatches resulting from DNA polymerase errors during replication.

Silent or synonymous mutations

Do not alter the amino acid encoded; these include many changes in the third nucleotide of a codon. Some silent mutations may, however, have serious consequences if they alter the splicing pattern of the gene.

DNA Repair

Due to consistent damage of DNA by the sources described above, maintenance of the genetic information requires constant repair of damaged DNA. All free living organisms have several mechanisms to repair damage to DNA. A key feature in most repair processes is the double-stranded nature of DNA, which allows restoration of the correct sequence on a damaged strand using the complementary genetic information on the other strand.

Excision repair

Excision repair is a general mechanism that can very accurately repair many different kinds of damage.

Frame-shifts

Frame-shifts usually lead to premature termination (or more rarely, elongation) of the encoded poly-peptide chain when stop codons are generated or removed by the frame-shift. Some chemicals can insert themselves between adjacent base pairs (know as inter-calating) in the DNA. This can lead to insertions or deletions of a single base pair, and thus a frame-shift.

Homologous recombination also occurs in the absence of DNA damage.

Homologous recombination also occurs in the absence of DNA damage as a mechanism to "shuffle" genes between a pair of homologous chromosomes that carry the same genes (i.e., each member of the chromosome 15 pair will shuffle with one another, but not with other chromosomes from a different pair). This happens during gamete (egg and sperm) formation as a way to generate genetic diversity on each pair of chromosomes before they are passed to the next generation. -------- Thus, two parents with various characteristics will likely see those characteristics "mixed up" in their children.

Frame-shift Mutations

Insertions or deletions of one or more base pairs (if the number of base pairs is not a multiple of 3) lead to frame-shift mutations that disrupt the coding of a protein.

DNA in cells

Is constantly being damaged by a variety of chemicals, both those resulting from normal cellular activity as well as environmental factors. Damage to DNA can block essential processes including DNA replication and transcription and can lead to mutations

What is the major difference between repair of DNA damage and repair of mis-matches?

It is in the choice of which base to remove.

MIs-Match Repair

Mis-match repair takes place soon after DNA replication to remove any replication errors. Mis-matches are not like DNA damage: there is no damaged or modified base present, just the wrong one of the four bases. The recognition of mis-matches relies on the distortion of the double-helical structure.

Point mutations.

Mutations that are changes of a single base pair are called point mutations. Several kinds of point mutations are possible, and point mutations can be categorized by their effect on a coding sequence.

Base excision repair (BER) and nucleotide excision repair (NER). The basic steps for both of these modes of repair include:

Recognize the damage Remove the damage by excising part of one strand to leave a gap Re-synthesize the sequence using genetic information from the other strand to fill the gap Ligate to restore continuity of the DNA backbone.

Nucelotide Excision Repair pathways

Repair damage that involves multiple nucleotides, such as thymine dimers.

Base Excision Repair pathways

Repair damaged, single bases in the DNA.

Chemotherapeutic agents attack DNA.

Some change the structure of individual bases while others, such as cisplatin, cross-link the two DNA strands together. The higher sensitivity of replicating cells to DNA damage means that rapidly dividing tumor cells are more sensitive to these damaging agents than are normal cells, but their damage to normal cells limits their dosing and use.

Cells with badly damaged DNA

Tend to be so impaired that they undergo apoptosis, or programmed cell death, to make room for their replacement by healthy cells.

Nucelotide excision repair

The best studied example of nucelotide excision repair is the repair of DNA damage caused by UV radiation. UV radiation can cause two thymines (Ts) that are adjacent to one another to fuse together to form a thymine dimer. This type of damage can only be removed by cutting away several nucleotides on each side of the lesion, so the damage is removed as a large oligonucleotide (Figure 1-22 part C).

Xeroderma pigmentosum (XP).

The left image shows the formation of thymine dimers as a result of UV damage. When the NER is functional, it repairs these dimers and the cells are fine. However, the defective NER pathway in XP means that UV damage, especially thymine dimers, cannot be effectively repaired and the damage accumulates in the DNA. The right image shows a child with XP whose sun-exposed skin cannot be effectively repaired. This leads to high rates of skin damage and skin cancer.

What are the major differences between the two kinds of repair?

The major differences are based on; how many nucleotides are removed. In base excision repair a single nucleotide is replaced (Figure 1-20), while in NER, ------ several nucleotides, usually around 30, are removed and replaced (Figure 1-21).

(See figure on page 3)

The top sequences show the normal DNA, mRNA, and protein sequences of hemoglobin. The bottom sequence demonstrate how a single mutation in the DNA can be propagated through the mRNA to the protein sequence. While not all changes to protein sequence lead to disease, this change in the hemoglobin protein sequence leads to sickle cell anemia.

How can mutations result?

They can result from replication errors, from damage to the DNA, or from errors introduced during repair of damage.

Non-Homologous End Joining.

This is a process in which damaged DNA with double-strand breaks is essentially stuck together in non-homologous ways, is a rather extreme and problematic repair pathway. Because the DNA is joined without regard to a similar sequence, this type of repair can lead to an increase in mutations at the damage site, as well as chromosomal "mix up". It is usually used under circumstances of extreme DNA damage as a last resort by the cell to save the DNA.

Xero-derma pigmen-tosum (XP).

This was the first disease demonstrated to be caused by defective DNA repair. Patients are photosensitive and highly susceptible to skin cancers in sun-exposed areas of the body; rates of skin cancer are 2000-5000 times higher than for the average person (Figure 1-13). XP is a rare disease that can be caused by defects in any of genes of the NER pathway, highlighting the importance of that pathway to repair UV damage, including thymine dimers.

Fact.

Translation of an mRNA does not have punctuation; rather, once the initiation codon is determined, successive triplets are read as codons. Therefore, whereas addition (or deletion) of a multiple of three base pairs in a coding region would add (or subtract) amino acids to (or from) a protein, the addition of other numbers of base pairs shifts the reading frame from that point onward.

Common sources of DNA damage include the following.

Ultraviolet (UV) radiation can cause adjacent T bases to fuse together to form a thymine dimer that blocks DNA replication and transcription. Ionizing radiation, such as X-rays and radioactive decay, can cause strand breaks as well as create reactive oxygen species that damage the bases. Chemicals that are carcinogens or mutagens alter the structure of DNA bases. Reactive oxygen species generated by processes in the cell can damage the DNA bases.

Non-sense mutations Fact:

Usually have more severe effects than mis-sense mutations because they lead to synthesis of truncated (and generally unstable) poly-peptides.

How does Homologous Recombination work?

When recombination takes place as a repair mechanism, the recombination machinery locates the DNA sequence that has homology (nearly identical sequence) to both sides of the lesion and then uses the sequence of the intact DNA strands to fix the double-strand break. The process results in some of the sequence from the broken strand being incorporated into the intact strand, and vice versa. The DNA from both strands essentially becomes mixed, but if the sequences were highly homologous (basically identical), then the gene sequences are not affected.

Fact

While normal individuals can still experience DNA damage from UV radiation that results in skin cancer, those with a damaged repair pathway cannot repair that damage efficiently and will accumulate more mutations at a faster rate. This increased rate of mutation accumulation results in a higher probability of developing skin cancer.