7.013 Recitation 6 – Spring 2018

7.013: Spring 2018: MIT 7.013 Recitation 6 – Spring 2018 (Note: The recitation summary should NOT be regarded as the substitute for lectures) Summary of Lectures 9 (2/26) and 10 (3/2): DNA replication: This occurs during the “S phase” (S = synthesis) of cell division when two doublestranded DNA molecules (dsDNA) are made from one dsDNA molecule. DNA replication is semiconservative and the newly synthesized strand is antiparallel and complementary to the template strand. Replication is continuous for one strand (“leading strand”) and discontinuous for the other (“lagging strand”). The lagging strand is made continuous by the action of the ligase enzyme. DNA polymerase catalyzes the addition of complementary deoxyribonucleotides (dNTPs) to the 3’ OH end of the newly synthesized strand. Replication starts at the ori site, which is usually an AT rich sequence. DNA polymerase is a primer dependent enzyme i.e. it adds complementary bases only to the 3’OH end of small RNA primer that are made by the primase enzyme in the cell. These RNA primers are later degraded by RNAses and the gaps sealed using the dNTPs and DNA polymerase. During replication, Helicase unwinds the two strands of DNA such that they can be replicated. The single stranded DNA binding proteins (SSDBP) bind to and stabilize the unwound/separated strands of DNA. Topoisomerases remove the extra coils that form as the new DNA is being synthesized and allow the DNA to unwind. Each replication cycle results in the shortening of chromosomal ends. This process is slowed down by the action of telomerase enzyme that adds telomere repeats to the chromosomal ends. Processes that repair mutations in DNA: DNA polymerase enzyme has a 3->5’ exonuclease activity due to which it proofreads each dNTP that it adds to increase the fidelity of replication. The fidelity of replication is further enhanced by mismatch repair and base - excision repair mechanism. These enzymes recognize the DNA strand with incorrect base(s) based on the difference in the methylation pattern of bases in the parental and newly synthesized DNA strand thus lowering the error rate to 1 in 109. Transcription: A gene is a sequence of bases that encodes a final product (mostly a protein but sometimes an RNA). It is defined as a promoter (a regulatory sequence to which the RNA polymerase and transcription factors bind) followed by the sequence of bases that are transcribed to RNA. Since the promoter is located prior to the transcribed region and the DNA strand that serves as the template for transcription is always read in a 3’->5’ direction to make an mRNA transcript in a 5’->3’ direction, it is the location of the promoter for a gene which determines which DNA strand (top or the bottom) serves as the template strand to make an RNA transcript. Each gene also has a transcription start site and a transcription stop site. The transcribed RNA can be one of three types: mRNA, tRNA or rRNA. All these types are identical in their chemical composition, but they differ in function. The mRNAs are transcribed from genes that encode proteins; these mRNAs get translated to proteins by ribosomes. The rRNAs and tRNAs participate in the process of translating mRNAs. Errors during transcription are far higher than errors during replication since the RNA polymerase unlike the DNA polymerase lacks the proofreading ability. But the cell tolerates transcription errors much better than errors during replication. It is also to be noted that RNA polymerase unlike DNA polymerase does not require a primer to make an RNA transcript. The rate of transcription is regulated at many levels: chromatin modifications, how strong the promoter is, the presence or absence of transcription factors and distal regulatory base sequences such as enhancers. 1