Digitally signed by Jason Raquin Roque DN: cn=Jason Raquin Roque, o, ou, email=jason_mike15@yahoo.com, c=PH Date: 2012.05.

15 14:07:18 +08'00'

Roque, Jason R. HUB32 DNA REPLICATION Introduction

Biochemistry - Lecture Mr. Gideon A. Legaspi

DNA replication is the process whereby an entire double-stranded DNA is copied to produce a second, identical DNA double helix. [1]

Figure 1. DNA Replication

DNA replication is a complicated process requiring the intensive action of many different proteins. The replication proteins are clustered or grouped together in the certain locations inside the cell. This may be termed as a small factory inside the living cell that manufactures DNA copies. The DNA to be replicated will undergo to the certain locations in the cell for the copying of DNA. The incoming DNA double helix is split into two single strands and each original single strand becomes half of a new DNA double helix. Because each resulting DNA double helix retains one strand of the original DNA, DNA replication is said to be semi-conservative. [1]

Figure 2. Schemes for DNA Replication

Mutation

Mutations, or heritable changes in the genetic material / DNA, may be gross (at the level of the chromosome) or point alterations (this technically means mutations not visible as cytological abnormalities and/or those which map to a single "point" in experimental crosses). [2]

Figure 3. DNA Mutation

Mutations are inheritable changes in the base sequence of nucleic acid -- the genetic material. An organism with these changes is called a mutant. Genetic recombination is the process where genes from two genomes are combined together. A mutant will be different from its parent, its genotype or genetic makeup has been altered. The phenotype or visible properties

of the mutant may or may not be altered. The genotype of a strain is indicated by use of three small italics letters followed by a capital letter and indicates the gene involved in a process (hisC indicates the gene for HisC protein). The phenotype of the strain is indicated by three letter code that ends in a +/-. An auxotroph is formed when a required nutritional material (amino acid for example) that the parent strain, prototroph, could make is no longer formed. [3]

Base pair (nucleotide pair) substitutions

These are of two types: transitions (purine to purine or pyrimidine to pyrimidine) and transversions (purine to pyrimidine or pyrimidine to purine).

The consequences of base substitution mutations in protein coding regions of a gene depend on the substitution and its location. They may be silent, not resulting in a new amino acid in the protein sequence, eg. GCA or GCG codons in mRNA both mean arginine [this is often true in the third position of a codon, especially with transitions because of "wobble" base pairing]. A base substitution could also result in an amino acid substitution; this is referred to as a missense mutation. For example, CTC in the DNA sense strand [GAG in mRNA] will specify a glutamate residue in the protein; this is altered to CAC in the DNA or GUG in the mRNA, resulting in a valine residue in the beta-globin protein chain causing sickle-cell anemia. Missense mutations may have very serious consquences, as in the case of sickle-cell anemia, mild consequences as in the case of hemoglobin C (a different amino acid substitution in position 6 of beta-globin) or no phenotype as in the case of two known amino acid substitutions at position 7 of betaglobin. Finally, base substitutions in a protein coding region may mutate an amino acid codon to a termination codon or vice versa. The former type, which results in a prematurely shortened protein is referred to as a nonsense mutation. The effects of nonsense mutations are variable depending upon how much of the truncated protein is present and is required for its function. [2]

Table 1. Codons in DNA [4]

Base substitution mutations may also occur in promoters or 5' regulatory regions of genes or in introns and may affect their transcription, translation, or splicing. Many of the beta-thalassemias are the result of these types of non-structural mutations that affect the level of expression of the globin genes. All of the types of mutation described above have been observed in human globin genes. Their consequences depend on what they do to the level of expression of the gene product and/or on what amino acid substitution may have occurred and where it is in the protein. [2]

Frameshift mutations

These result from the insertion or deletion of one or more (not in multiples of three) nucleotides in the coding region of a gene. This causes an alteration of the reading frame: since codons are groups of three nucleotides, there are three possible reading frames for each gene although only one is used. [2]

A mutation of this sort changes all the amino acids downstream and is very likely to create a nonfunctional product since it may differ greatly from the normal protein. Further, reading frames other than the correct one often contain stop codons which will truncate the mutant protein prematurely. [2]

Elongation

During the elongation phase of DNA replication, both DNA strands are simultaneously copied. To accomplish this feat, a large multi-subunit protein complex, composed of two DNA polymerases, a helicase, and several accessory proteins, assembles at the border of the unzipped DNA molecule, called the replication fork. Using the 3-OH of the RNA primers (hybridized to each original DNA strand), DNA polymerase adds complementary deoxynucleotide triphosphates (dNTPs) as directed by each DNA template strand. As DNA polymerase adds subsequent dNTPs, the associated helicase continues to open the DNA molecule to provide access to the template strands. [5]

Figure 4. Chain Elongation reaction catalyzed by DNA polymerase

Termination Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular way. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes, but ends at the telomere region of repetitive DNA close to the end. This shortens the telomere of the daughter DNA strand. This is a normal process in somatic cells. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. [6]

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E coliregulate this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome. [6]

Figure 5. Termination of DNA through Tus protein.

DNA TRANSCRIPTION

Introduction

Transcription is the first stage of the expression of genes into proteins. In transcription, a

mRNA (messenger RNA) intermediate is transcribed from one of the strands of the DNA molecule. The RNA is called messenger RNA because it carries the 'message' or genetic information from the DNA to the ribosomes, where the information is used to make proteins. RNA and DNA use complementary coding, where base pairs match up, similar to how the strands of DNA bind to form a double helix. One difference between DNA and RNA is that RNA uses uracil in place of the thymine used in DNA. RNA polymerase mediates the manufacture of an RNA strand that complements the DNA strand. RNA is synthesized in the 5' > 3' direction (as seen from the growing RNA transcript). There are some proofreading mechanisms for transcription, but not as many as for DNA replication. Sometimes coding errors occur. [7]

Figure 6. DNA Transcription Initiation

The initiation of transcription in bacteria begins with the binding of RNA polymerase to the promoter in DNA. Transcription initiation is more complex in eukaryotes, where a group of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. [8]

Figure 7. Initiation of Transcription

Elongation

One strand of DNA serves as the template for RNA synthesis, but multiple rounds of transcription may occur so that many copies of a gene may be produced. [9]

Figure 8. Elongation of Transcription

Termination

Termination is the final step of transcription. Termination results in the release of the newly synthesized mRNA from the elongation complex. [10]

Figure 9. Termination of Transcription

References

1. Retrieved from http://www.wiley.com/college/pratt/0471393878/student/animations/ dna_replication/index.html on September 23, 2011 2. Retrieved from http://www-personal.ksu.edu/~bethmont/mutdes.html on September 23, 2011 3. Retrieved from http://cwx.prenhall.com/brock/chapter9/objectives/deluxe-content.html on September 23, 2011 4. Retrieved from http://www.chemguide.co.uk/organicprops/aminoacids/dna6.html on September 23, 2011 5. Retrieved from http://www.nature.com/scitable/popular-discussion/885 on September 23, 2011 6. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK21113/#8156 on September 23, 2011 7. Retrieved from http://chemistry.about.com/od/biochemistry/ss/transcription.htm on September 23, 2011 8. Retrieved from http://chemistry.about.com/od/biochemistry/ss/transcription_4.htm on September 23, 2011 9. Retrieved from http://chemistry.about.com/od/biochemistry/ss/transcription_6.htm on September 23, 2011 10. Retrieved from http://chemistry.about.com/od/biochemistry/ss/transcription_7.htm on September 23, 2011