Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs.
DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000648:ultrastructure*; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism*; D012617:Sea Urchins; D014357:Trypsin / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D014357:Trypsin