DNA repair

syllabus 1 -Introduction

2-Rpair mechanisms 3-DNA damage check points

4-Regulation 5 -Hereditary DNA repair disorders

Introduction DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome.

Sources of damage:1-loss of a bases resulting in

apurinic/apyrimidinic (AP) sites (abasic sites).2-base modifications, such as alkylations or

deamidations which converts cytosine, adenine and guanine to uracil .

3-Replication errors and base conversions can generate mismatch nucleotide pairs

4-Failures in normal DNA metabolism by topoisomerases and nuclease or ionizing radiation can generate single-strand and double-strand breaks.

5-Photodamage by uv light can generate pyrimidine dimers, such cyclobutane pyrimidine dimers (CPDs)Chemical agents and reactive oxygen species (ROS) can modify bases.

N.Bs :Majority of DNA damage affect the 1ry structure of

double helix .

DNA repair is dependent on many factors: - cell type - age of the cell -extracellular

environment A cell that has accumulated a large amount of DNA damage can enter one of three possible states:

1-an irreversible state of dormancy, known as senescence

2-cell suicide, also known as apoptosis 3-unregulated cell division, which can lead to

cancer

DNA Damage & Mutation DAMAGE MUTATION

1-Damages are physical abnormalities in the DNA

2-DNA damages can be recognized by enzymes, and, thus, they can be correctly repaired.

3-If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein will also be blocked

1-A mutation is a change in the base sequence of the DNA.

2-A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be repaired.

3-Mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates

REPAIR MECHANISMS In addition to DNA polymerase 3’à5’

exonuclease(the DNA Pol III has proofreading capabilities that correct replication mistakes by means of exonuclease activity working 3'->5') ..Mammalian cells utilize TWO major DNA repair pathways:

- single strand damage -db strand breaks

Reverse Excision Non Homo Homo

BER NER MMR

A-SS damage : 1- reversal ( direct reversal ): These mechanisms do not require a template,

since the types of damage they counteract can occur in only one of the four bases.(this type repaired without removing abase or nucleotide)E.X :

1-The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the

enzyme photolyase.

2 -Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT) .

2-Excision : In which the damaged base or bases are removed and then replaced with

the correct ones. a-Base excision repair:

DNA's bases may be modified by deamination or alkylation.  the DNA glycosylase can recognize the damaged site and remove its base forming AP site ( Apurinic/ Apyrimidinic).  Then, the AP endonuclease removes the AP site and neighboring nucleotides.  The gap is filled by DNA polymerase I and DNA ligase .

N.B: -Each DNA glycosylase is generally specific for one type of lesion.

_Humans have at least four types glycosylase with different specifictices .

B- Nucleotide excision repair: NER differs from BER in several ways:

-It uses different enzymes.-Even though there may be only a single "bad"

base to correct, its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage.

-In NER a multisubunit enzyme hydrolyzes two phosphodiester bonds one on either side of the distorsion caused by lesion ( in human it hydrolyzes the 6th bond on 3\ side & the 22 bond on the 5\ end producing a fragment of 27-29 nucleotides ) resulting in gap filled by DNA polymerase1 & finally DNA ligase seals

the nick .

C- Mismatch repair: To repair mismatched bases, the system has to

know which base is the correct one.  In E. coli, this is achieved by a special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC sequences.  Immediately after DNA replication, the template strand has been methylated, but the newly synthesized strand is not methylated yet.  Thus, the template strand and the new strand can be

distinguished .

B- Db strand damage : There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule :

1-Direct joining: of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ).

Errors in direct joining may be a cause of the various translocations that are associated with cancers .

2-Homologous Recombination:. Here the broken ends are repaired using the

information on the intact sister chromatid (available in G2 after chromosome duplication), or on the homologous chromosome.

Two primary models :1-DSBR pathway (sometimes called the double

Holliday junction model) 2 -the synthesis-dependent strand annealing

(SDSA) pathway .

N.Bs:Whether homologous recombination or NHEJ is used to repair double-strand breaks is largely determined by

the phase of cell cycle.Homologous recombination repairs DNA before the cell enters mitosis (M phase). It occurs during and shortly after DNA replication, in the S and G2 phases of the cell cycle, when sister chromatids are more easily available.While NHEJ is predominant in the G1 phase of the cell cycle, when the cell is growing but not yet ready to divide.

Cyclin- dependent kinases( CDKs ,) which modify the activity of other proteins by adding phosphate g roups

) , to that is phosphorylating( , t hem are important regulators of homologous recombination in eukaryotes .

DNA damage check points

The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis (&the common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division ).

-After DNA damage, cell cycle checkpoints are activated Checkpoint activation pauses the cell cycle and gives the cell time to repair the

damage before continuing to divide .

-DNA damage checkpoints occur at the G1/S and G2/M boundaries. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,[31] whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade,eventually leading to cell cycle arrest.

-53P is an important downstream target of ATM and ATR, as it is required for inducing apoptosis following DNA damage.[33] At the G1/S checkpoint, p53 functions by deactivating the CDK2/cyclin E complex. Similarly, p21 mediates the G2/M checkpoint by deactivating the CDK1/cyclin B complex.

-To get an idea of just the first layer of complexity in these systems, let's assume there is some damage to DNA, such as a single-strand break. Here is the process that would happen:

1-DNA damage is detected by sensor proteins: PAR activation this occurs within seconds of damage detection. PARP 1 and PARP2 are activated by single strand breaks and double strand breaks.

2-ATM activation (ATM and ATR kinases as seem to send out the distress signal to recruit the right DNA repair proteins. They do this by phosphorylating mediator proteins.)

3-MDCI recruitment (This induces a signaling cascade.)

4-RNF8 recruitment5-RNFi68 recruitment

6-BRCA1 and 53BP1 recruitment (Numbers 4-6 are particular proteins that are

recruited in a very specific order to do specific activities to repair a single strand break).

7-Based on certain factors such as timing and order of recruitment, any one of these results may happen: the cell cycle could be delayed, DNA could be repaired by replacing nucleotide bases, differentiation may be halted (senescence), cell death (apoptosis), transcription and splicing controls, or metabolic regulation.

Hereditary DNA repair disordersDefects in the NER mechanism are responsible for several genetic disorders, including:

Xeroderma pigmentosum :hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging

Cockayne syndrome :hypersensitivity to UV and chemical agents

Trichothiodystrophy :sensitive skin, brittle hair and nailsMental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:' Werner s syndrome :premature aging and

retarded growth' Bloom s syndrome :sunlight hypersensitivity,

high incidence of malignancies (especially leukemias).

Ataxia telangiectasia :sensitivity to ionizing radiation and some chemical agents.

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