DNA Replication PDF

During DNA replication, base pairing enables existing DNA strands to serve as templates for new complimentary strands 1  When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complimentary strand.   Nucleotides line up along the template strand...

During DNA replication, base pairing enables existing DNA strands to serve as templates for new complimentary strands 1  When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complimentary strand.   Nucleotides line up along the template strand according to the base-pairing rules. The nucleotides are linked to form new strands (complementary). DNA Replication: 1. During DNA replication, 2base pairing enables existing DNA strands to serve as templates for new complimentary strands. 2. Several enzymes and other proteins carry out DNA replication: Helicase, Primase, Polymerase, Ligase. The ends of DNA molecules are replicated by a special mechanism. The Replication Mechanism 3  It takes E. coli less than an hour to copy each of the 5 million base pairs in its single chromosome and divide to form two identical daughter cells.  A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours.  This process is remarkably accurate, with only one error per billion nucleotides.  A helicase; untwists and separates the template DNA strands at the replication fork.  Single-strand binding proteins; keep the unpaired template strands apart during replication. • The replication of a DNA molecule begins at special site called origin of replication which is a single specific sequence of nucleotides that is recognized by the replication enzymes. • Replication enzymes separate the strands, forming a replication “bubble”. – Replication proceeds in both directions until the entire molecule is copied.  In eukaryotes, there may be hundreds or thousands of bubbles (each has origin sites for replication) per chromosome.   At the origin sites, the DNA strands separate forming a replication “bubble” with replication forks at each end. The replication bubbles elongate as the DNA is replicated and eventually fuse.  Primer: (a short segment of RNA, 10 nucleotides long) is required to start a new chain. 5  Primase: (an RNA polymerase) links ribonucleotides that are complementary to the DNA template into the primer. • DNA polymerases: catalyze the elongation of new DNA at a replication fork. After formation of the primer, DNA polymerases can add deoxyribonucleotides to the 3’ end of the ribonucleotide chain. • Another DNA polymerase later replaces the primer ribonucleotides with deoxyribonucleotides complimentary to the template.  DNA polymerases can only add nucleotides to the free 3’ end of a growing DNA strand.  A new DNA strand can only elongate in the 5’->3’ direction.  At the replication fork, one parental strand (3’-> 5’ into the fork), the leading strand, can be used by polymerases as a template for6 a continuous complimentary strand. To elongate the other new strand of DNA in the obligatory 5’->3’ direction, DNA pol III must work along the other template strand in the direction away from the replication Fork . The DNA strand elongating in this direction is called the lagging strand. the lagging strand is synthesized discontinuously, as a series of segments (called Okazaki fragments. laying  Okazaki fragments (each about 100-200 nucleotides) are joined by DNA ligase to form the sugar-phosphate backbone of a single DNA strand. Step 1  Helicases: Enzymes that separate the DNA strands  Helicase move along the strands and breaks the hydrogen bonds between the complimentary nitrogen bases  Replication Fork: the Y shaped region that results from the separation of the strands Step 2 • DNA Polymerase: enzymes that add complimentary nucleotides. • Nucleotides are found floating freely inside the nucleus • Covalent bonds form between the phosphate group of one nucleotide and the deoxyribose of another • Hydrogen bonds form between the complimentary nitrogen bases Step 3 • DNA polymerases finish replicating the DNA and fall off. • The result is two identical DNA molecules that are ready to move to new cells in cell division. • Semi-Conservative Replication: this type of replication where one strand is from the original molecule and the other strand is new  Each strand is making its own new strand.  DNA synthesis is occurring in two different directions  One strand is being made towards the replication fork and the other is being made away from the fork. The strand being made away from the fork has gaps.  Gaps are later joined by another enzyme, DNA ligase • • The strands in the double helix are antiparallel. The sugar-phosphate backbones run in opposite directions. – Each DNA strand has a 3’ end with a free OH group attached to deoxyribose and a 5’ end with a free phosphate group attached to deoxyribose. – The 5’ -> 3’ direction of one strand runs counter to ‫ ُمعاكس لـ‬the 3’ -> 5’ direction of the other strand. SUMMARY OF DNA REPLICATION MECHANISM The two DNA-strands separate forming replication bubbles. Each strand functions as a template for synthesizing new complementary & lagging strands via primers, polymerase and ligase. 3 5 T A C T G A C A T G A C T G 3 5 Complementary (leading) strand T A C T G Primer Polymeras Ligase e A C 5 3 Lagging strand (complementary) Okazaki fragments Templates 1 2 3 4 10 Fig. 16.15, Page 298 Types of RNAs • mRNA: is the carrier of the genetic “message” from the DNA to the cytosol. • rRNA: is the major component of ribosomes. • tRNA: is the carrier of specific amino acids from the cytosol to ribosimes thus help to form polypeptides. RNA transcription and translation are the two main processing that link gene to protein • The information content of DNA is in the form of specific sequences of nucleotides along the DNA strands. • The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins. • Proteins are the links between genotype and phenotype. – For example, Mendel’s dwarf pea plants lack a functioning copy of the gene that specifies the synthesis of gibberellins (which stimulate the normal elongation of stems). • Genes provide the instructions for making specific proteins. • The bridge between DNA and protein synthesis is RNA. • The specific sequence of hundreds or thousands of nucleotides in each gene carries the information for the primary structure of a protein, the linear order of the 20 possible amino acids.  During RNA transcription, a DNA strand provides a template for the synthesis of a complementary RNA strand.  Transcription of a gene produces a messenger RNA (mRNA) molecule.  During RNA translation (at ribosomes), the information contained in the order of nucleotides in mRNA is used to determine the amino acid sequence of a polypeptide.  The basic mechanics of transcription and translation are similar in eukaryotes and prokaryotes.  Because bacteria lack nuclei, transcription and translation are coupled.  Ribosomes attach to the leading end of a mRNA molecule while transcription is still in progress. • In a eukaryotic cell, all transcription occurs in the nucleus and translation occurs mainly at ribosomes in the cytoplasm. • In addition, before the primary transcript can leave the nucleus it is modified in various ways during RNA processing before the finished mRNA go to the cytoplasm. • To summarize, genes program protein synthesis via genetic messenger RNA. • The molecular chain of command in a cell is : DNA Transcription mRNA Translation Protein In the genetic code, nucleotide triplets specify amino acids • • • • • • Triplets of nucleotide bases are the smallest units that can code for all the amino acid. In the triplet code three consecutive bases specify an amino acid. The genetic instructions for a polypeptide chain are written in DNA as a series of three-nucleotide words (triplets). During transcription, one DNA strand (the template strand) provides an RNA template. The complementary RNA molecule is synthesized according to base-pairing rules, except that uracil is the complementary base to adenine. During translation, blocks of three nucleotide bases (codons), are decoded ‫ فك الشفرة‬into a sequence of amino acids. • read in the 5’->3’ direction along the mRNA. • The codon UUU coded for the amino acid phenylalanine. • The codon AUG not only codes for the amino acid methionine but also indicates the start of translation. • A specific codon indicates a specific corresponding amino acid, but the amino acid may be the translation of several possible codons. The reading frame and subsequent codons are read in groups of three nucleotide bases (codon). Ay • stop start A During translation, the codons are • In summary, genetic information is encoded as a sequence of base triplets (codons) which is translated into a specific amino acid during protein synthesis. A  Transcription can be separated into three stages: 1- initiation 2- elongation, 3- termination.  Promotor contains the starting point for the transcription of a gene.  Promotor also includes a binding site for RNA polymerase.  Thus, RNA- polymerase can recognize and bind directly to the promotor region. 17  As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time.  The enzyme adds nucleotides to the 3’ end of the growing strand.  Behind the point of RNA synthesis, the double helix reforms and the RNA molecule moves away.  Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA. Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm. 1)At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap which function as: 1) 2) protect mRNA from hydrolytic ‫ ُمحلل‬enzymes. a translation start point for ribosomes. Eukaryotic cells modify RNA after transcription 2) At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail. The poly(A) tail facilitate the export of mRNA from the nucleus. 3) The removal of large portions of the RNA molecule that is initially synthesized (This cut-and-paste job, called RNA splicing) Translations is the RNA-directed synthesis of a polypeptide 21  In the process of translation, a cell sends a series of codons along a mRNA molecule.  Transfer RNA (tRNA) transfers amino acids from the cytoplasm to a ribosome.  The ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain.  During translation, each type of tRNA links a mRNA codon with the appropriate amino acid.  Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodon, at the other end.  The anticodon base-pairs with a complementary codon on mRNA.  If the codon on mRNA is UUU, a tRNA with an AAA anticodon and carrying phenyalanine will bind to it.  Codon by codon, tRNAs deposit amino acids in the prescribed order and the ribosome joins them into a polypeptide chain. 22 Fig. 17.12, Page 314 The Structure and Function of Transfer RNA    tRNA molecules: are transcribed from DNA in the nucleus. Once it reaches the cytoplasm, each tRNA is used repeatedly for the following functions:1) to pick up its relevant amino acid in the cytosol, 2) to deposit the amino acid at the ribosome 3) to return to the cytosol to pick up another copy of that amino acid. The anticodons of some tRNAs recognize more than one codon. Figure 17.15 The structure of transfer RNA (tRNA).  Ribosomes: the protein making machine facilitate the coupling of the tRNA anticodons with mRNA codons.   Each ribosome has a large and a small subunit formed in the nucleolus. Ribosome is composed of proteins and ribosomal RNA (rRNA), the most abundant RNA in the cell.  rRNA is transcribed in the nucleus, then bind to special proteins to form the ribosomal subunits in the nucleolus.  The subunits exit the nucleus via nuclear pores.  The large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule. Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules. 25 1) 2) 3) The P site holds the tRNA carrying the growing polypeptide chain. The A site carries the tRNA with the next amino acid. The E site at which the discharged tRNA leave the ribosome.  Translation occurs in three stages: 1- initiation of translation 2- elongation of polypeptide chain 3- termination of translation 26 1. Initiation: brings together mRNA, a tRNA (with the first amino acid) and the two ribosomal subunits (large & small).   First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon. Initiation factors bring in the large subunit such that the initiator tRNA occupies the P site. Methionin e Guanosine triphosphate Fig. 17.17, Page 317 2. Elongation: Consists of a series of three step cycles as each amino acid is added to the proceeding one in 3 steps:- a) 27 Codon recognition, an elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresponding anticodon of tRNA carrying the appropriate amino acid [This step requires the hydrolysis of two guanosine triphosphate (GTP)]. b) Peptide bond formation: an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site. This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site. c) Translocation of tRNA: the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.  The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed. exit 3.   Termination: Occurs when one of the three stop codons reaches the A site. A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site. This frees the polypeptide and the translation complex disassembles. Fig. 17.19 The free and bound ribosomes are both active participants in protein synthesis. Polyribosomes : • A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide. • Multiple ribosomes, polyribosomes, may trail along the same mRNA. • Thus, a single mRNA is used to make many copies of a polypeptide simultaneously. Mutations: They affect protein structure and function • Mutations are changes in the genetic material of a cell (or virus). • These include large-scale mutations in which long segments of DNA are affected (e.g., translocations, duplications, and inversions). I.Point mutation : is a chemical change in just one base pair of a gene. • If these occur in cells producing gametes, they may be transmitted to future generations (offspring). For e.g., sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein. O O O 31 A)- Base-pair substitution • It is a point mutation that results in replacement of a pair of complimentary nucleotides with another nucleotide pair. a) Silent mutation: some base-pair substitutions (point mutation) have little or no impact on protein function which lead to switches from one amino acid to another with similar properties. Other base-pair substitutions cause a detectable change in a protein which impact function. b) Missense mutations: a point mutation that still code for an amino acid but change the resulting protein. c) Nonsense mutations: change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein. 32 B)- Insertions and deletions • It is a mutation in which additions or losses of nucleotide pairs occurs and causes Frameshift mutation. – These have a disastrous effect on the resulting protein • When frameshift occurs, all the nucleotides downstream of the deletion or insertion will be improperly grouped into new codons. – The result will be extensive missense, ending sooner or later in nonsense - premature termination. 33 • Mutations can occur in a number of ways. – – – • Errors can occur during DNA replication, DNA repair, or DNA recombination. These can lead to base-pair substitutions, insertions, or deletions, as well as mutations affecting longer stretches of DNA. These are called spontaneous mutations. Mutagens : are chemical or physical agents that interact with DNA to cause mutations. 1. Physical agents include high-energy radiation like X-rays and ultraviolet light. 2. Chemical agents may operate in several ways. – – – Some chemicals are base analogues that may be substituted into DNA, but that pair incorrectly during DNA replication. Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix. Still others cause chemical changes in bases that change their pairing 34 properties.