DNA Repair Mechanisms part 01

 

 # What is DNA Damage?

DNA damage is an alteration in the chemical structure of DNA, such as a break in a strand of DNA, a base missing from the backbone of DNA, or a chemically changed base such as 8-OHdG. Damage to DNA that occurs naturally can result from metabolic and hydrolytic process.

# Cause & Consequences of DNA damage:

Cause:

¨       Despite an efficient repair system for the damaged DNA, replication errors do accumulate that ultimately result in mutation. The human body possesses 10¹⁴ nucleated cells, each with 3 x 10⁹ base pairs of DNA. It is estimated that about 10¹⁶ cell divisions occur in a lifetime. If 10^{-1 0} mutations per base pair per cell generation escape repair, this results in about one mutation per 10⁶ base pairs in genome.

¨       Besides the possible errors in replication, the DNA is constantly subjected to attack by both physical and chemical agents. These include radiation, free radicals, chemicals etc., which also result in mutations.

Consequences:

       It is fortunate that a great majority of the mutations probably occur in the DNA that does not encode proteins, and consequently will not have any serious impact on the organism. This is not, however, all the time true, since mutations do occur in the coding regions of DNA also. There are situations in which the change in a single base pair in the human genome can cause a serious disease e.g. sickle cell anemia.

# Types of DNA Damage:

The damages done to DNA by physical, chemical and environmental agents may be broadly classified into four categories with different types (Table 24.1).

Single base alteration: The occurrence of spontaneous       deamination bases in aqueous solution at 37℃ is well known. Cytosine gets deaminated to form uracil while adenine forms hypoxanthine.

Spontaneous depurination, due to cleavage of glycosyl bonds (that connect purines to the backbone) also occurs. It is estimated that 2000-1 0,000 purines may be lost per mammalian cell in 24 hours. The depurinated sites are called as abasic sites. Originally, they were detected in purines, and called apurinic sites (AP sites) which represent lack of purine. Now, the term AP sites is generally used to represent any base lacking in DNA.

Two-base alterations: The production of reactive oxygen species is often associated with alteration of bases e.g. formation of 8-hydroxy guanine.

Ultraviolet radiations result in the formation of covalent links between adjacent pyrimidines along the DNA strand to form pyrimidine dimers. DNA chain breaks can be caused by ionizing radiations (e.g. X - rays).

Chain breaks: Free radical formation and oxidative damage to DNA increases with advancement of age.

MUTATIONS

Mutation: refers to a change in the DNA structure of a gene. The substances (chemicals) which can induce mutations are collectively known as mutagens.

The changes that occur in DNA on mutation are reflected in replication, transcription and translation.

# Types of mutations:

Mutations are mainly of two major types   (Fig; 24 .l4).

®     point mutations

®     frameshift mutations

1.    Point mutations: The replacement of one base pair by another results in point mutation. They are of two sub-types.

a)       Transitions: In this case, a purine (or a pyrimidine) is replaced by another.

b)      Transversions: These are characterized by replacement of a purine by a pyrimidine or vice versa.

2.       Frameshift mutations: These occur when one or more base pairs are inserted in or deleted from the DNA, respectively, causing insertion or deletion mutations.

# Consequence of Point Mutation:

    The change in a single base sequence in point mutation may cause one of the following (Fig.24.15)

1.  Silent mutation: The codon (of mRNA) containing the changed base may code for the same amino acid. For instance, UCA codes for serine and change in the third base (UCU) still codes for serine. This is due to degeneracy of the genetic code. Therefore, there are no detectable effects in silent mutation.

  2. Missense mutation: In this case, the changed base may code for a different amino acid. For example, UCA codes for serine while ACA codes for threonine. The mistaken (or missense) amino acid may be acceptable, partially acceptable or unacceptable with regard to the function of protein molecule. Sickle-cell anemia is a classical example of missense mutation.

 3. Nonsense mutation: Sometimes, the codon with the altered base may become a termination (or nonsense) codon. For instance, change in the second base of serine codon (UCA) may result in UAA. The altered codon acts as a stop signal and causes termination of protein synthesis, at that Point.

# Consequences of Frameshift Mutation:

The insertion or deletion of a base in a gene results in an altered reading frame of the mRNA (hence the name frameshift). The machinery of mRNA (containing codons) does not recognize that a base was missing or a new base was added. Since there are no punctuations in the reading of codons, translation continues. The result is that the protein synthesized will have several altered amino acids and/or prematurely terminated protein.

# ORF (Open Reading Frame) & Identification of ORF in a genome:

In molecular genetics, an open reading frame (ORF) is the part of a reading frame that has the potential to code for a protein or peptide. An ORF is a continuous stretch of codons that do not contain a stop codon (usually UAA, UAG or UGA). An AUG (start codon) codon within the ORF (not necessarily the first) may indicate where translation starts. The transcription termination site is located after the ORF, beyond the translation stop codon, because if transcription were to cease before the stop codon, an incomplete protein would be made during translation. In eukaryotic genes with multiple exons, ORFs may span exons. These would be spliced into an ORF in the mRNA.

Gene finding in organism specially prokaryotes starts form searching for an open reading frames (ORF). An ORF is a sequence of DNA that starts with start codon “ATG” (not always) and ends with any of the three termination codons (TAA, TAG, TGA).

Biological significance/Characteristics of ORF:

®     One common use of open reading frames is as one piece of evidence to assist in gene prediction.

®     Long ORFs are often used, along with other evidence, to initially identify candidate protein coding regions in a DNA sequence.

®     The presence of an ORF does not necessarily mean that the region is ever translated. For example, in a randomly generated DNA sequence with an equal percentage of each nucleotide, a stop-codon would be expected once every 21 codons.

®     A simple gene prediction algorithm for prokaryotes might look for a start codon followed by an open reading frame that is long enough to encode a typical protein, where the codon usage of that region matches the frequency characteristic for the given organism's coding regions. By itself even a long open reading frame is not conclusive evidence for the presence of a gene.

®     On the other hand, it has been proven that some short ORFs (sORFs) that lack the classical hallmarks of protein-coding genes (both from ncRNAs and mRNAs) can produce functional peptides.

ORF finding tools:

ORF Finder: The ORF Finder (Open Reading Frame Finder) is a graphical analysis tool which finds all open reading frames of a selectable minimum size in a user's sequence or in a sequence already in the database. This tool identifies all open reading frames using the standard or alternative genetic codes. The deduced amino acid sequence can be saved in various formats and searched against the sequence database using the BLAST server. The ORF Finder should be helpful in preparing complete and accurate sequence submissions. It is also packaged with the Sequin sequence submission software. (sequence analyser)

ORF Investigator: ORF Investigator is a program which not only gives information about the coding and noncoding sequences but also can perform pairwise global alignment of different gene/DNA regions sequences. The tool efficiently finds the ORFs for corresponding amino acid sequences and converts them into their single letter amino acid code, and provides their locations in the sequence. The pairwise global alignment between the sequences makes it convenient to detect the different mutations, including single nucleotide polymorphism. Needleman and Wunsch algorithms are used for the gene alignment. The ORF Investigator is written in the portable Perl programming language, and is therefore available to users of all common operating systems.

ORFPredictor:  OrfPredictor is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with a hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments, otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences. The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where the coding region begins and ends. OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects.

       

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